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COMPARATIVE EVALUATION OF INCLUSION COMPLEX OF
ACECLOFENAC PREPARED BY DIFFERENT TECHNIQUES
A Dissertation Submitted to
The Tamil Nadu Dr. M.G.R. Medical University
Chennai - 600 032
In partial fulfillment for the award of Degree of
MASTER OF PHARMACY
(Pharmaceutics)
Submitted by
P.SATHISH KUMAR
Register No. 26106008
Under the Guidance of
Mr. T. AYYAPPAN, M. Pharm.
Assistant Professor
Department of Pharmaceutics
ADHIPARASAKTHI COLLEGE OF PHARMACY
(Accredited by “NAAC” with a CGPA of 2 .74 on a four point scale at “B” Grade)
MELMARUVATHUR - 603 319
MAY 2012
CERTIFICATE
This is to certify that the dissertation entitled “COMPARATIVE EVALUATION
OF INCLUSION COMPLEX OF ACECLOFENAC PREPARED BY
DIFFERENT TECHNIQUES” Submitted to The Tamil Nadu Dr. M.G.R. Medical
University, Chennai, in partial fulfillment for the award of the Degree of the Master of
Pharmacy was carried out by P.SATHISH KUMAR (Register No. 26106008) in the
Department of Pharmaceutics under my direct guidance and supervision during the
academic year 2011-2012.
T. AYYAPPAN, M. Pharm.,
Assistant Professor,
Department of Pharmaceutics,
Place: Melmaruvathur Adhiparasakthi College of Pharmacy,
Date: Melmaruvathur - 603 319.
CERTIFICATE
This is to certify that the dissertation entitled “COMPARATIVE
EVALUATION OF INCLUSION COMPLEX OF ACECLOFENAC
PREPARED BY DIFFERENT TECHNIQUES” is the bonafide research work
carried out by P.SATHISH KUMAR (Register No. 26106008) in the Department
of Pharmaceutics, Adhiparasakthi College of Pharmacy, Melmaruvathur which is
affiliated to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, under the
guidance of Mr. T. AYYAPPAN, M. Pharm., Assistant Professor, Department of
Pharmaceutics, Adhiparasakthi College of Pharmacy, during the academic
year 2011-2012.
Prof. Dr. T. VETRICHELVAN, M.Pharm., Ph.D.,
Principal,
Place: Melmaruvathur Adhiparasakthi College of Pharmacy,
Date: Melmaruvathur - 603 319.
My Heartfelt Dedication To
My Beloved Parents,
&
My beloved ones...""
ACKNOWLEDGEMENT
First and foremost, I wish to express my deep sense of gratitude to his
Holiness ARULTHIRU AMMA, President, ACMEC Trust, Melmaruvathur for their
ever growing blessings in each step of the study.
With great respect and honor, I extend my sincere thanks to THIRUMATHI
LAKSHMI BANGARU ADIGALAR, Vice President, ACMEC Trust,
Melmaruvathur for given me an opportunity and encouragement all the way in
completing the study.
I got inward bound and brainwave to endure experimental investigations
in novel drug delivery systems, to this extent; I concede my inmost special gratitude
and thanks to Mr. T. AYYAPPAN, M.Pharm., Assistant Professor, Department of
Pharmaceutics, Adhiparasakthi College of Pharmacy for the active guidance, valuable
suggestions and a source of inspiration where the real treasure of my work.
I owe my sincere thanks with bounteous pleasure to
Prof. Dr. T.VETRICHELVAN, M.Pharm., Ph.D., Principal, Adhiparasakthi
College of Pharmacy, without his encouragement and supervision it would have been
absolutely impossible to bring out the work in this manner.
I have great pleasure in express my sincere heartfelt thanks to
Prof. K. SUNDARA MOORTHY, B.Sc., M.Pharm., Dr. S. SHANMUGAM,
M. Pharm., Ph.D., Professor, Department of Pharmaceutics, for encouragement and
support for the successful completion of this work.
My sincere thanks to our lab technicians Mrs. S. KARPAGAVALLI,
D.Pharm., and Mr. M. GOMATHI SHANKAR, D.Pharm., for their kind help
throughout this work.
I am very grateful to our Librarian Mr. SURESH, M.L.I.S., for his kind
cooperation and help in providing all reference books and journals for the completion
of this project.
I am highly indebted to Tristar Pharmaceuticals, Pondicherry, for generous
gift sample of the drug. I thank to Mrs. M. Sujitha, I -M.Pharm., for kind obligation
in procuring gift sample of Aceclofenac.
I also thank to Alkem Laboratories, Mumbai, for generous gift sample of the
drug carrier. I thank to Mr. Zanjurne Amol Hanamant, M. Phram., for her kind
obligation in procuring gift sample of β - Cyclodextrin.
I am thankful to my dear friends and seniors, for being a great source of help
whenever I needed and for sharing their ideas and extending support during the course
of study.
Last but not the least, I can hardly find any words enough to express gratitude
to my father M. PICHAIPILLAI, my mother P. VIJAYA, my brothers
P.JAYAPRAKASH, P. CHANDRAMOHAN, my sister P.PRIYARAJA, and all
my relatives for their frequent prayer, tremendous encouragement, support and love
which has proved to be a real source of inspiration, and will remain so far the life to
come, without which it would have been impossible for me to achieve this success.
P.SATHISH KUMAR
CONTENTS
CHAPTER
TITLE
PAGE No.
1.
INTRODUCTION
1
2.
LITERATURE SURVEY
2.1. Literature Review
32
2.2. Drug Profile
42
2.3. β-Cyclodextrin Profile
46
3. AIM AND OBJECTIVE
48
4.
PLAN OF WORK
50
5.
MATERIALS AND EQUIPMENTS
5.1. Materials Used
52
5.2.Equipments Used
53
6.
PREFORMULATION STUDIES
6.1.Identification of Drug
54
7.
FORMULATION OF INCLUSION COMPLEXES
57
7.1. Methods of Preparation of Inclusion Complexes
58
8. EVALUATION OF INCLUSION COMPLEXES
8.1. Phase Solubility Studies
59
8.2. UV-Spectrometric Study of Prepared Complexes
60
8.3. Fourier Transform Infrared (FTIR) Spectroscopy
60
8.4. Differential Scanning Calorimetry(DSC) Analysis
60
8.5. X-Ray Diffraction Studies
61
CHAPTER
TITLE
PAGE No.
8.6. Drug Content Estimation
61
8.7. In vitro Dissolution Studies
61
8.8. Stability Studies
62
9.
RESULTS AND DISCUSSION
9.1. Identification of Drug
63
9.2. Phase Solubility Studies
70
9.3. UV-Spectrometric Study of Prepared Complexes
71
9.4. Fourier Transform Infrared (FTIR) Spectroscopy
72
9.5. Differential Scanning Calorimetry(DSC) Analysis
74
9.6. X-Ray Diffraction Studies
76
9.7. Drug Content Estimation
78
9.8. In vitro Dissolution Studies
79
9.9. Stability Studies
92
10.
SUMMARY AND CONCLUSION
97
11.
FUTURE PROSPECTS
100
12.
BIBLIOGRAPHY
101
13.
JOURNAL PUBLICATION
LIST OF TABLES
TABLE No.
CONTENTS
PAGE No.
1.1
Characteristics of cyclodextrins
20
1.2
Stability requirement for maintenance of shelf life
28
5.1
List of raw materials with source
52
5.2 List of equipments with model/make
53
7.1
Composition of inclusion complexes of Aceclofenac
57
8.1 Parameters were used for the dissolution study
62
9.1
Solubility of Aceclofenac in various solvents
63
9.2
Percentage loss on drying of Aceclofenac
64
9.3
Characteristic frequencies in FTIR spectrum of
Aceclofenac
65
9.4 Data of concentration and absorbance for Aceclofenac
in Methanol 66
9.5
Data for calibration curve parameter of Methanol 67
9.6
Data of concentration and absorbance for Aceclofenac
in Distilled water
68
9.7
Data for calibration curve parameter of Distilled water
69
9.8
Assay of Aceclofenac
69
9.9
Phase solubility studies of Aceclofenac-β-cyclodextrin
complexes
70
9.10
Interpretation of FTIR spectrum of Aceclofenac
73
9.11
Data for DSC thermogram parameters
75
9.12
Drug content estimation of Aceclofenac inclusion
complexes
78
9.13
Dissolution profile of formulation F1
79
9.14
Dissolution profile of formulation F2
80
9.15
Dissolution profile of formulation F3
81
TABLE No.
CONTENTS
PAGE No.
9.16
Dissolution profile of formulation F4
82
9.17
Dissolution profile of formulation F5
83
9.18
Dissolution profile of formulation F6
84
9.19
Dissolution profile of formulation F7
85
9.20
Dissolution profile of formulation F8
86
9.21
Dissolution profile of formulation F9
87
9.22
Comparative dissolution profile of all formulations
90
9.23 Stability study of formulation F2 of inclusion complex
Aceclofenac at room temperature 25° C ± 2° C / 60 %
RH ± 5 % and accelerated temperature 40° C ± 2° C /
75 % RH ± 5 %.
92
LIST OF FIGURES
FIGURE No.
CONTENTS
PAGE No.
1.1
Structure of cyclodextrins
18
1.2
Molecular structure of β-cyclodextrin
19
1.3
Complexation of cyclodextrin
22
2.1
Structure of Aceclofenac
42
2.2
Pathway describing general anti-inflammatory action of
Aceclofenac
43
2.3
Structure of β-cyclodextrin
46
9.1
FTIR spectrum of Aceclofenac
64
9.2
λ max of Aceclofenac in Methanol
65
9.3
λ max of Aceclofenac in Distilled Water
66
9.4
Standard graph of Aceclofenac in Methanol
67
9.5
Standard graph of Aceclofenac in Distilled Water
68
9.6
Phase solubility studies of Aceclofenac-β-cyclodextrin
complexes
70
9.7
λ max of prepared complexes
71
9.8
FTIR spectrum of Aceclofenac
72
9.9
FTIR spectrum of β-cyclodextrin
72
9.10
FTIR spectrum of formulation F2
73
9.11
DSC thermogram of Aceclofenac
74
9.12
DSC thermogram of β-cyclodextrin
74
9.13
DSC thermogram of Formulation F2
75
9.14
X-ray diffraction of pure Aceclofenac
76
9.15
X-ray diffraction of β-cyclodextrin
76
9.16
X-ray diffraction of formulation F2
77
9.17
Dissolution profile of formulation F1
79
9.18
Dissolution profile of formulation F2
80
9.19
Dissolution profile of formulation F3
81
FIGURE No.
CONTENTS
PAGE No.
9.20
Dissolution profile of formulation F4
82
9.21
Dissolution profile of formulation F5
83
9.22
Dissolution profile of formulation F6
84
9.23
Dissolution profile of formulation F7
85
9.24
Dissolution profile of formulation F8
86
9.25
Dissolution profile of formulation F9
87
9.26 Dissolution profile of inclusion complex (kneading method)
containing various ratio i.e. 1:1, 1:2, 1:3
88
9.27 Dissolution profile of inclusion complex (common solvent
method) containing various ratio i.e. 1:1, 1:2, 1:3
88
9.28 Dissolution profile of inclusion complex (physical mixture)
containing various ratio i.e. 1:1, 1:2, 1:3
89
9.29
Comparative dissolution profile of all formulations
90
9.30
Comparative dissolution profile of drug content, marketed
formulation and formulation F2
91
9.31 Comparisons of drug content before and after stability
period at room temperature (25° C ± 2° C / 60 % RH ± 5 %)
93
9.32
Comparisons of in vitro cumulative % drug release before
and after stability period at room temperature (25° C ± 2° C /
60 % RH ± 5 %).
93
9.33 Comparisons of drug content before and after stability
period at accelerated temperature (40° C ± 2° C / 75 % RH
± 5 %)
94
9.34 Comparisons of in vitro cumulative % drug release before
and after stability period at accelerated temperature (40° C ± 2° C / 75 % RH ± 5 %)
94
9.35
Comparisons of drug content before and after stability
period at room temperature and accelerated temperature
95
9.36 Comparisons of in vitro cumulative % drug release before
and after stability period at room temperature and
accelerated temperature.
95
ABBREVIATIONS
% ---- Percentage
β ---- Beta
γ ---- Gamma
°C ---- Degree Celsius
µ ---- Micro
µg ---- Microgram
ACE ---- Aceclofenac
BCS ---- Biopharmaceutics Classification System
CD ---- Cyclodextrin
cm ---- Centimeter
DE ---- Dissolution Efficiency
DSC ---- Differential Scanning Calorimetry
edn ---- Edition
Fig ---- Figure
FTIR ---- Fourier Transform Infrared Spectroscopy
GIT ---- Gastrointestinal Tract
gm ---- Grams
ICH ---- International Conference on Harmonization
IP ---- Indian Pharmacopoeia
KBr ---- Potassium Bromide
MDT ---- Mean Deviation Time
mg ---- Milligram
min ---- Minute ml
---- Millilitre mM --
-- Millimoles nm -
--- Nanometer
NSAID ---- Non- steroidal anti inflammatory drug
RH ---- Relative Humidity
RPM ---- Revolutions Per Minute
S.No. ---- Serial Number
SD ---- Standard Deviation
t ---- Time
UV ---- Ultra Violet
vol ---- Volume
w/v ---- Weight/Volume
w/w ---- Weight/Weight
XRD ---- X-Ray Diffraction
α ---- Alpha
β-CD ---- Betacyclodextrin
λmax ---- Absorption Maximum
INTRODUCTION
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 1
1. INTRODUCTION
(Raymond C.Rowe., 2003)
Cyclodextrins are cyclic oligosaccharides containing at least six D-(+)-
glucopyranose units attached by α (1Æ4) glucoside bonds. The three natural
cyclodextrins, α, β, and γ differ in their ring size and solubility. They contain 6, 7 or 8
glucose units, respectively.
Cyclodextrins occur as white, practically odourless, fine crystalline powders,
having a slightly taste. Some cyclodextrins derivatives occur as amorphous powders.
Cyclodextrins were discovered approximately 100 years ago with the foundation of
cyclodextrin chemistry being laid down in the first half of this century. In the beginning,
only small amounts of relatively impure cyclodextrins could be generated and high
production costs prevented their industrial usage. Recent bio-technological advancements
have resulted in drastic improvements in the efficiency of manufacture of cyclodextrins,
lowering the cost of these materials and making highly purified cyclodextrins and
cyclodextrin derivatives available.
Cyclodextrins are used to increase the solubility of water insoluble drugs, through
inclusion complexation. Natural cyclodextrins have been used extensively for this
purpose. However, they are characterized by relatively low solubility in water, which
limits their application. Hence, chemically modified cyclodextrins are gaining
considerable interest to improve the physicochemical properties of cyclodextrins.
Cyclodextrins are known to form an inclusion complex with many drugs of appropriate
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 2
molecular size and polarity in hydrophobic drug molecules. The resulting complex
generally leads to an improvement in some of the properties of drugs in terms of
solubility, bioavailability and tolerability.
Poorly water soluble drugs are generally associated with slow drug absorption
leading eventually to inadequate and variable bioavailability.
Cyclodextrins are ‘bucket like’ or ‘cone like’ toroid molecules, with a rigid
structure and central cavity, the size of which varies according to the cyclodextrin type.
The internal surface of the cavity is hydrophobic and the outside of the torus is
hydrophilic; this is due to arrangement of hydroxyl groups within the molecule. This
arrangement permits the cyclodextrin to accommodate a guest molecule within the cavity,
forming an inclusion complex.
The interaction of guest molecules with cyclodextrins may induce useful
modifications of the chemical and physical properties of guest molecules, which may
lead to improve stability, solubility in aqueous medium and bioavailability. Poorly water
soluble drugs therefore can be orally administered in the complex form by taking
advantage of the well established low toxicity of the cyclodextrins by the oral route. β-
Cyclodextrin appears to be the most useful complexing agent because of its unique cavity
size, and ease with which it can be obtained on the industrial scale leading to reasonably
cheaper price of the compound. Pharmaceutical applications of cyclodextrins as additive
and drug complexing agents have been rapidly growing.
The behavior of the drug in the body involves the fate of the drug during it’s
transmit as well as its fate while in the biophase. The drug potentially interacts with a
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 3
variety of substances leading to undesired drug loss as loss desired drug of absorption. In
every instance the degree of this loss or absorption is a function of the drug and the type
of the dosage form.
1.1. Biopharmaceutics and Pharmacokinetics:
(Brahmankar D.M and Sunil B. Jaiswal., 2009)
Biopharmaceutics is the study of physiologic and pharmaceutical factors
influencing drug release and absorption from dosage forms. The optimal delivery of the
active moiety to the site of action depends on an understanding of specific interaction
between formulation variables and biological variables. Absorption is defined as the
amount of drug that reaches the general circulation in unchanged form from the site of
administration.
The drug efficacy can be severely limited by poor aqueous solubility. An
insoluble or sparingly soluble drug, if administered the rate of absorption and extent of
bioavailability is controlled by dissolution rate in gastrointestinal fluid. The therapeutic
effectiveness of a drug whose absorption is dissolution rate limited is hampered by its
poor dissolution in an aqueous medium. The absorption of solid drugs administered
orally can be depicted by the following chart.
Solid drugs Kd Drug solution Ka Drug in systemic
in GI fluids Dissolution in GI fluids Absorption circulation
where Kd and Ka are the rate constants for dissolution and absorption process
respectively.
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 4
1.2. Methods for Enhancement of Bioavailability:
(Brahmankar D.M and Sunil B. Jaiswal., 2009)
As far as the definition of bioavailability is concerned, a drug with poor
bioavailability is the one with
1. Poor aqueous solubility and / or slow dissolution rate in the biological fluids.
2. Poor stability of the dissolved drug at the physiologic pH.
3. Inadequate partition coefficient and thus poor permeation through the
biomembrane.
4. Extensive presystemic metabolism.
The three major approaches in overcoming the bioavailability problems due to
such causes are:
a. The Pharmaceutic Approach which involves modification of formulation,
manufacturing process or the physicochemical properties of the drug without
changing the chemical structure.
b. The pharmacokinetic approach in which the pharmacokinetics of the drug is
altered by modifying its chemical structure.
c. The Biologic approach whereby the route of drug administration may be changed
such as changing from oral to parenteral route.
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 5
The second approach of chemical structure modification has a number of
drawbacks of being very expensive and time consuming, requires repetition of clinical
studies and long time for regulatory approval. Moreover, the new chemical entity may
suffer from another pharmacokinetic disorder or bear the risk of precipitating adverse
effects. Only the pharmacokinetic approach will be dealt herewith.
The attempts, whether optimizing the formulation, manufacturing process or
physicochemical properties of the drug, are mainly aimed at enhancement of dissolution
rate as it is the major rate limiting step in the absorption of most drugs. There are several
ways in which the dissolution rate of a drug can be enhanced. Some of the widely used
methods, most of which are aimed at increasing the effective surface area of the dugs will
be discussed briefly.
1.2.1. Techniques of Solubility Enhancement:
(Mohanachandran P.S et al., 2010; Brahmankar D.M and Sunil B. Jaiswal., 2009)
There are various techniques available to improve the solubility of poorly soluble
drugs. Some of the approaches to improve the solubility are,
a) Physical Modifications:
Particle size reduction:
• Micronization
• Nanosuspension
• Sonocrystalisation
• Supercritical fluid process
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 6
Modification of the crystal habit:
• Polymorphs
• Pseudopolymorphs
Drug dispersion in carriers:
• Eutectic mixtures
• Solid dispersions
• Solid solutions
Complexation:
• Use of complexing agents
Solubilization by surfactants:
• Microemulsion
• Self microemulsifying drug delivery systems
b) Chemical Modifications:
Physical Modifications:
Particle size reduction: Particle size reduction can be achieved by micronisation and
nanosuspension. Each technique utilizes different equipments for reduction of the
particle size.
Micronization: The solubility of drug is often intrinsically related to drug particle
size. By reducing the particle size, the increased surface area improves the
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 7
dissolution properties of the drug. Conventional methods of particle size reduction,
such as communication and spray drying, rely upon mechanical stress to disaggregate
the active compound.
Nanosuspension: Nanosuspensions are sub-micron colloidal dispersion of pure
particles of drug, which are stabilized by surfactants. The advantages offered by
nanosuspension is increased dissolution rate is due to larger surface are exposed,
while absence of Ostwald ripening is due to the uniform and narrow particle size
range obtained, which eliminates the concentration gradient factor.
Sonocrystallisation: Recrystallization of poorly soluble materials using liquid
solvents and antisolvents has also been employed successfully to reduce particle size.
The novel approach for particle size reduction on the basis of crystallization by using
ultrasound is Sonocrystallisation. Sonocrystallisation utilizes ultrasound power
characterized by a frequency range of 20-100 kHz for inducing crystallization. It’s
not only enhances the nucleation rate but also an effective means of size reduction
and controlling size distribution of the active pharmaceutical ingredients. Most
applications use ultrasound in the range 20 kHz-5 MHz.
Modification of the crystal habit: Polymorphism is the ability of an element or
compound to crystallize in more than one crystalline form. Different polymorphs of
drugs are chemically identical, but they exhibit different physicochemical properties
including solubility, melting point, density, texture and stability. Broadly polymorphs
can be classified as enantiotropes and monotropes based on thermodynamic
properties.
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 8
In the case of an enantiotropic system, one polymorphs form can change
reversibly into another at a definite transition temperature below the melting point,
while no reversible transition is possible for monotropes. Once the drug has been
characterized under one of this category, further study involves the detection of
metastable form of crystal. Metastable forms are associated with higher energy and
thus higher solubility.
Drug dispersion in carriers : The solid dispersion approach to reduce particle size
and therefore increase the dissolution rate and abso rption of drugs was first
recognized in 1961. The term “solid dispersions” refers to the dispersion of one or
more active ingredients in an inert carrier in a solid state, frequently prepared by the
melting method, solvent method, or fusion solvent- method. Novel additional
preparation techniques have included rapid precipitation by freeze drying and using
supercritical fluids and spray drying, often in the presence of amorphous hydrophilic
polymers and also using methods such as melt extrusion.
Complexation: Complexation is the association between two or more molecules to
form a nonbonded entity with a well defined stoichiometry. Complexation relies on
relatively weak forces such as London forces, hydrogen bonding and hydrophobic
interactions.
Staching complexation: Staching complexes are formed by the overlap of the planar
regions of aromatic molecules, Nonpolar moieties tend to be squeezed out of water by
the strong hydrogen bonding interactions of water. This causes some molecules to
minimize the contact with water by aggregation of their hydrocarbon moieties. This
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 9
aggregation is favored by large planar nonpolar regions in the molecule. Stached
complexes can by homogeneous or mixed. The former is known as self association
and latter as complexation.
Inclusion complexation: Inclusion complexes are formed by the insertion of the
nonpolar molecule or the nonpolar region of one molecule (known as guest) into the
cavity of another molecule or group of molecules (known as host). The major
structural requirement for inclusion complexation is a snug fit of the guest into the
cavity of host molecule. The cavity of host must be large enough to accommodate the
guest and small enough to eliminate water, so that the total contact between the water
and the nonpolar regions of the host and the guest is reduced. Three naturally
occurring CDs are α-Cyclodextrin, β -Cyclodextrin, and γ – Cyclodextrin. The
complexation with cyclodextrins is used for enhancement of solubility. Cyclodextrin
inclusion is a molecular phenomenon in which usually only one guest molecule
interacts with the cavity of a cyclodextrin molecule to become entrapped and form a
stable association. The internal surface of cavity is hydrophobic and external is
hydrophilic; this is due to the arrangement of hydroxyl group within the molecule.
The kinetics of cyclodextrin inclusion complexation has been usually analyzed in
terms of a one – step reaction or a consecutive two – step reaction involving
intracomplex structural transformation as a second step. Cyclodextrins is to enhance
aqueous solubility of drugs through inclusion complexation. It was found that
cyclodextrins increased the paclitaxel solubility by 950 fold, complex formation of
rofecoxib, celecoxib, clofibrate, melarsoprol, taxol, cyclosporine A etc., with
cyclodextrins improves the solutility of particular drugs.
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 10
Approaches for Making Inclusion Complexes:
Physical blending method: A solid physical mixture of drug and CDs are prepared
simply by mechanical trituration. In laboratory scale CDs and drug are mixed
together thoroughly by trituration in a mortar and passes through appropriate sieve to
get the desired particle size in the final product.
Kneading method: This method is based on impregnating the CDs with little amount
of water or hydroalcoholic solutions to converted into a paste. The drug is then added
to the above paste and kneaded for a specified time. The kneaded mixture is then
dried and passed through sieve if required.
Co – precipitation technique: This method involves the co – precipitation of drug
and CDs in a complex. In this method, required amount of drug is added to the
solution of CDs. The system is kept under magnetic agitation with controlled process
parameters and the content is protected from the light. The formed precipitate is
separated by vacuum filtration and dried at room temperature in order to avoid the
loss of the structure water from the inclusion complex.
Solution / solvent evaporation method: This method involves dissolving of the drug
and CDs separately in to two mutually miscible solvents, mixing of both solutions to
get molecular dispersion of drug and complexing agents and finally evaporating the
solvent under vacuum to obtain solid powdered inclusion compound. Generally, the
aqueous solution of CDs is simply added to the alcoholic solution of drugs. The
resulting mixture is stirred for 24 hours and evaporated under vacuum at 45° C. The
dried mass was pulverized and passed through a 60 – mesh sieve. This method is
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 11
quite simple and economic both on laboratory and large scale production and is
considered alternative to the spray drying technique.
Neutralization precipitation method: This method is based on the precipitation of
inclusion compounds by neutralization technique and consists of dissolving the drug
in alkaline solutions like sodium / ammonium hydroxide and mixing with an aqueous
solution of CDs. The resultant clear solution is then neutralized under agitation
using HCI solution till reaching the equivalence point. A white precipitate is being
formed at this moment, corresponding to the formation of the inclusion compound.
This precipitate is filtered and dried.
Milling / Co – grinding technique: A solid binary inclusion compounds can be
prepared by grinding and milling of the drug and CDs with the help of mechanical
devices. Drug and CDs are mixed intimately and the physical mixture is introduced
in an oscillatory mill and grinded for suitable time. Alternatively, the ball milling
process can also be utilized for preparation of the drug – CD binary system. The ball
mill containing balls of varied size is operated at a specified speed for a
predetermined time, and then it is unloaded, sieved through a 60 – mesh sieve. This
technique is superior to other approaches from economic as well as environmental
stand point in that unlike similar methods it does not require any toxic organic
solvents. This method differs from the physical mixture method where simple
blending is sufficient and in co-grinding it requires to achieve extensive combined
attrition and impact effect on powder blend.
Inclusion Complexes of Aceclofenac Introduction
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 12
Atomization / spray drying method: Spray – drying is a common technique used in
pharmaceuticals to produce a dry powder from a liquid phase. Another application is
its use as a preservation method, increasing the storage stability due to the water
elimination. This method represents one of the most employed methods to produce
the inclusion complex starting from a solution. The mixture pass to a fast elimination
system propitiate solvent and shows a high efficiency in forming complex. Besides,
the product obtained by this method yield the particles in the controlled manner
which in turn improves the dissolution rate of drug in complex form.
Lyophilization / Freeze drying technique: In order to get a porous, amorphous
powder with high degree of interaction between drug & CD, lyophilization / freeze
drying technique is considered as a suitable. In this technique, the solvent system
from the solution is eliminated through a primary freezing and subsequent drying of
the solution containing both drug & CD at reduced pressure. Thermolabile
substances can be successfully made into complex form by this method. The
limitations of the technique are long time process and yield poor flowing powdered
product. Lyophilization /freeze drying technique are considered as an alternative to
solvent evaporation and involve molecular mixing of drug and carrier in a common
solvent.
Microwave irradiation method: This technique involves the microwave irradiation
reaction between drug and complexing agent using a microwave oven. The drug and
CD in definite molar ratio are dissolved in a mixture of water and organic solvent in a
specified proportion into a round bottom flask. The mixture is reacted for short time
of about one to two minutes at 60° C in the microwave oven. After the reaction
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completes, adequate amount of solvent mixture is added to the above reaction mixture
to remove the residual, uncomplexed free drug and CD. The precipitate so obtained
is separated using whatman filter paper, and dried in vacuum oven at 40° C from
48 hrs.
Supercritical antisolvent technique: In this technique, carbon dioxide is used as
anti- solvent for the solute but as a solvent with respect to the organic solvent. The
use of supercritical carbon dioxide is advantageous as its low critical temperature and
pressure makes it attractive for processing heat – labile pharmaceuticals. It is also non
– toxic, nonflammable, inexpensive and is much easier to remove from the polymeric
materials when the process is complete, even through small amount of carbon dioxide
remains trapped inside the polymer, it poses no danger to the consumer. Supercritical
particle generation processes are new and efficient route for improving bioavailability
of pharmaceutically active compounds. In addition, supercritical fluid processes were
recently proposed as a new alternative method for the preparation of drug
cyclodextrin complexes. Supercritical carbon dioxide is suggested as a new
complexation medium due to its properties of improved mass transfer and increased
solvating power. This method constitutes one of the most innovators methods to
prepare the inclusion complex of drug with CD in solid state. This is a non – toxic
method as it is not utilizing any organic solvent, fast process, maintenance cost is low
with promising results, but it requires a quite high initial cost.
In this technique, first, drug and CD are dissolved in a good solvent then the
solution is fed into a pressure vessel under supercritical conditions, through a nozzle
(i.e. sprayed into supercritical fluid anti – solvent). When the solution is sprayed into
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supercritical fluid anti – solvent, the anti – solvent rapidly diffuses into that liquid
solvent as the carrier liquid solvent counter diffuses into the anti– solvent. Because
of the supercritical fluid expanded solvent has lower solvent power than the pure
solvent, the mixture becomes supersaturated resulting in the precipitation of the solute
and the solvent is carried away with the supercritical fluid flow.
Solubilization by surfactants:
Surfactants are molecules with distinct polar and nonpolar regions. Most
surfactants consist of a hydrocarbon segment connected to a polar group. The polar
group can be anionic, cationic, zwitterionic or nonionic. When small apolar molecules
are added they can accumulate in the hydrophobic core of the micelles.
This process of solubilization is very important in industrial and biological
processes. The presence of surfactants may lower the surface tension and increase the
solubility of the drug within an organic solvent.
Micro emulsions: The term microemulsion was first used by Jack H. Shulman in
1959. A microemulsion is a four component system composed of external phase,
internal phase, surfactant and cosurfactant. The addition of surfactant, which is
predominately soluble in the internal phase unlike the cosurfactant, results in the
formation of an optically clear, isotropic, thermodynamically stable emulsion. It is
termed as microemulsion because of the internal or dispersed phase is <0.1 µ droplet
diameter. The formation of microemulsion is spontaneous and does not involve the
input of external energy as in case of coarse emulsions. The surfactant and the
cosurfactant alternate each other and form a mixed film at the interface, which
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contributes to the stability of the microemulsions. Non – ionic surfactants, such as
Tweens and Labrafil with high hyrophile – lipophile balances are often used to ensure
immediate formation of oil – in – water droplets during production. Advantages of
microemulsion over coarse emulsion include its ease of preparation due to
spontaneous formation, thermodynamic stability, transparent and elegant appearance,
increased drug loading, enhanced penetration through the biological membranes,
increased bioavailability, and less inter-and intra – individual variability in drug
pharmacokinetics.
Chemical Modifications:
For organic solutes that are ionizable, changing the pH of the system may be
simplest and most effective means of increasing aqueous solubility. Under the proper
conditions, the solubility of an ionizable drug can increase exponentially by adjusting
the pH of the solution. A drug that can be efficiently solubilized by pH control should
be either weak acid with a low pKa or a weak base with a high pKa. Similar to the
lack of effect of heat on the solubility of non – polar substances, there is little effect
of pH on nonionizable substances. Nonionizable, hydrophobic substances can have
improved solubility by changing the dielectric constant of the solvent by the use of
co-solvents rather than the pH of the solvent. The use of salt forms is a well known
technique to enhanced dissolution profiles. Salt formation is the most common and
effective method of increasing solubility and dissolution rates of acidic and basic
drugs. An alkaloid base is, generally, slightly soluble in water, but if the pH of
medium is reduced by addition of acid, and the solubility of the base is increased as
the pH continues to be reduced. The reason for this increase in solubility is that the
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base is converted to a salt, which is relatively soluble in water. The solubility of
slightly soluble acid increased as the pH is increased by addition of alkali, the reason
being that a salt is formed.
1.3. Monomolecular Inclusion Complexes:
(Martin’s., 2006)
Monomolecular inclusion compounds involve the entrapment of a single guest
molecule in the cavity of one host molecule. Most of the host molecules are
cyclodextrins. Cyclodextrins are cyclic oligo-saccharides containing a minimum of
six D-(+) glucopyranose units attached by alpha-1,4 linkage. Three types of
cyclodextrins namely α, β, γ consist of 6, 7 and 8 units of glucose, respectively.
Alpha cyclodextrin has the smallest cavity (internal diameter almost 5 A). Beta and
gamma cyclodextrins have larger internal diameter (6 and 8 A. respectively) and are
useful for pharmaceutical technology.
The structures of cyclodextrins assume a truncated cone and can
accommodate a wide varity of compounds. The interior of the cavity is relatively
hydrophobic, whereas the entrance of the cavity is hydrophilic in nature.
Applications : These types of complexes are extensively studied for their possible
uses in the design of dosage forms.
Enhanced solubility: The solubility of retinonic acid (0.5 mg/litre) is increased to
160 mg/litre by complexation with β -cyclodextrin.
Enhanced dissolution: The dissolution rate of drugs such as famotidine and
tolbutamide is enhanced by complexation with β -cyclodextrin.
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Enhanced stability: The stability of drugs such as aspirin, benzocaine, ephedrine and
testosterone is improved when complexed with β cyclodextrin. The inclusion
complexes protect the drugs by preventing the exposure of the functional groups to
the exterior of the environment.
Sustained release: Ethylated β -cyclodextrin retards the release of drugs such as
diltiazem and isosorbide dinitrate, when complexed. The drug releases slowly for
prolonged periods and provides sustained effect.
1.4. Cyclodextrin:
(Loftsson T. et al., 1996; Gerold Mosher et al., 2007; Szejtli J.,1990)
Cyclodextrin or cycloamylases which have recently been recognized as useful
pharmaceutical excipient and comprise a family of cyclic oligosaccharides produced
from starch by enzymatic degradation. The enzyme cyclodextrin-glucosyl transferase
produced by Bacillus macerans acts on partially hydrolyzed starch and produces a
mixture of cyclic and acyclic dextrins from which cyclodextrin are isolated.
The β-cyclodextrin appears to be the most useful complexing agent because of
its unique cavity size and ease with which it can be obtained on industrial scale. β-
Cyclodextrin displays a water solubility of 1.8 g/100 ml. Recently derivatives of β-
cyclodextrin have been received considerable attention because of their higher water
solubility (>50 g/100 ml). Partial methylation of some of the hydroxyl (OH) groups in
cyclodextrin reduces the inter-molecular hydrogen bonding, leaving some hydroxyl
groups free to interact with water, thus increasing the aqueous solubility of
cyclodextrins. They belong to methylated β-cyclodextrins (dimethyl β-CD and
trimethyl β-CD). Hydroxy alkylation of the hydroxyl groups of the cyclodextrin
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produced hydroxy alkylated β-cyclodextrins (2 hydroxy ethyl β-CD, 2 hydroxypropyl
β-CD). This modification results in greater solubility, of hydroxypropyl β-
cyclodextrin and its complexes compared to β-cyclodextrin. The hydroxy groups and
the hydroxypropyl groups are on the exterior of the molecule and interact with water
to provide the increased aqueous solubility of the hydroxypropyl β-cyclodextrin.
Fig. 1.1: Structure of cyclodextrins
The most stable three-dimensional structure of cyclodextrin is a toroid with
the larger and smaller openings presenting hydroxyl groups to the external
environment and mostly hydrophobic functionality lining the interior and the cavity.
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Fig. 1.2: Molecular structure of β-cyclodextrins
It is the unique configuration that gives cyclodextrins their interesting
properties and creates the thermodynamic driving force needed to form host guest
complexes with a polar molecules and functional groups.
1.4.1. Chemistry of Cyclodextrin:
(Raymond C.Rowe., 2003)
Cyclodextrin are cyclic, non-reducing, water-soluble oligosaccharides. Three
different forms of cyclodextrin known are α, β and γ. Cyclodextrins are also called
Schardinger dextrins, cycloglucans or cycloamylases are α-1, 4 linked cyclic
oligosaccharides obtained from enzymatic conversion of starch. The parent or natural
cyclodextrins contain 6, 7 or 8 glucopyranose units.
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Table 1.1: Characteristics of cyclodextrins
α β γ
No. of glucose units 6 7 8
Molecular weight 972 1135 1297
Cavity diameter 4.7 – 5.3 6.0 – 6.5 7.5 – 8.3
Water solubility (g/100 ml) 14.50 1.85 23.2
Content (dry basis) >98 % >98 % >98 %
Specific rotation in aqueous
solution (α)
D, 20
+ 147 to +150°
+160 to +164°
+174 to +180°
Water <10 % <14 % <11 %
Heavy metals <5 ppm <5 ppm <5 ppm
Cavity height (A°) 7.9 7.9 7.9
Cavity volume (A°)³ 174 262 427
In addition, all three cyclodextrins exhibit good flow properties and handling
characteristics and are:
• Thermally stable (<200° C)
• Using stable alkaline solution (pH < 14).
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• Stable in acidic solution (pp>3)
• Biocompatible.
1.4.2. Method of Manufacture:
Cyclodextrins are manufactured by the enzymatic degradation of starch using
specialized bacteria (Bacillus macerans). The insoluble β-CD organic solvent
complex is separated from the non-cyclic starch, and the organic solvent removed in
vacuole so that less than 1 ppm of solvent remains in the β-CD. The cyclodextrin is
then carbon treated and crystallized from water, dried and collected.
Hydroxyethyl-β-CD is made by reacting β-CD with ethylene oxide, while
hydroxypropyl-β-CD is made by reacting with propylene oxide.
1.4.3. Complexation of Cyclodextrins:
(Martin’s., 2006)
The ability to form inclusion compounds in aqueous solution is due to the
typical arrangement of glucose units in aqueous solutions. Cyclodextrins form
complexes with many drugs through a process in which the water molecules located
in the central cavity are replaced by either the whole drug molecule or more
frequently by some lipophilic portion of the drug structure.
The interior of the cyclodextrin cavity is relatively hydrophobic because of the
presence of the skeletal carbons and ethereal oxygen, which comprise the cavity,
whereas the cavity entrances are hydrophilic owing to the presence of the primary and
secondary hydroxyl groups. As the water molecules located inside the cavity cannot
satisfy their hydrogen bonding potential, they are having high enthalpy than bulk
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water molecules located in the solution. Water inside the cavity tends to be squeezed
out and to be replaced by more hydrophobic species. Thus, molecules of appropriate
size and stereochemistry can be included in the cyclodextrin by hydrophobic
interactions.
Water is preferred solvent for complexation. The guest or proton of the which
complexes with the cavity of cyclodextrin as a non-polar (hydrophobic) and prefers
the non-polar environment of the cavity of cyclodextrin rather than polar aqueous
environment as a result water provides a driving force for complexation reaction in
addition to dissolving the guest and cyclodextrin.
Fig. 1.3: Complexation of cyclodextrin
The above figure provides a schematic representation of the equilibrium
involved in forming an inclusion complex between cyclodextrin and toluene in the
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presence of a small amount of water. In general, there are four energetically favorable
interactions that help shift the equilibrium to right:
• The displacement of polar water molecules from the a polar cyclodextrin
cavity.
• The increased number of hydrogen bonds formed as the displaced water
returns to the larger pool.
• A reduction of the repulsive interactions between the hydrophobic guest and
the aqueous environment.
• An increase in the hydrophobic interactions as the guest inserts itself into the a
polar cyclodextrin cavity.
While this initial equilibrium to form the complex is very rapid (often within
minutes), the final equilibrium can take much longer to reach. Once inside the
cyclodextrin cavity, the guest molecule makes conformational adjustments to take
maximum advantage of the weak Vander Waals’ forces that exist.
1.5. Preparation of Complexes:
(Sanoferjan A.M et al., 2000; Chowdary K.P.R et al., 2000)
Cyclodextrin complexes are prepared by the following methods:
a) Kneading method
b) Common solvent method
c) Physical mixture
d) Co-precipitate method
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a) Kneading method:
β-Cyclodextrin was taken in a glass mortar and little water was added and
mixed to obtain a homogenous paste. Drug was then added slowly while grinding.
The mixture was ground for 1 h during this process appropriate quantity of water was
added to maintain suitable consistency. The paste was dried in an oven at 40° C for
48 hr. The dried complex was taken for study.
b) Common solvent method:
Drug and β-Cyclodextrin were dissolved in 25 % ammonia and the solvent
was allowed to evaporate overnight at room temperature. The complex so prepared
was pulverized and sifted through sieve no.80.
c) Physical mixture:
Previously weighted drug and Cyclodextrin mixture was blended in glass mortar
for about an hour and passed through sieve no.85 to get physical mixture and stored
in desiccator over fused calcium chloride.
d) Co-precipitate Method:
Drug and cyclodextrin in different molar ratio are dissolved in different solvent.
The solution of the drug is incorporated into solution of cyclodextrin drop wise with
continuous stirring at room temperature for 1 hour and then slowly evaporated on a
boiling water bath. The inclusion complex precipitated as a crystalline powder, which
is then pulverized, sieved and stored in a desiccator till free from any traces of the
organic solvent.
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1.6. Phase Solubility Technique: (Higuchi T. and Connors K.A., 1965)
“T. Higuchi and K.A. Connors” developed the phase solubility technique. It is
based on research related to how complexes of different complexing agents such as
cyclodextrin, caffeine, polyvinyl pyrrolidone and some aromatic acids affect the
aqueous solubility of drug.
1.6.1. Phase Solubility Diagrams:
Phase solubility diagrams are plots of drug solubility versus cyclodextrin
concentration. The stoichiometry of drug/cyclodextrin complexes and the numerical
values of their stability constants are frequently obtained from phase solubility
diagrams.
The physicochemical properties of free drug molecules are different from
those bound to the cyclodextrin molecules. Likewise, the physicochemical properties
of free cyclodextrin molecules are different from those in the complex. In theory, any
methodology that can be used to observe these changes in addition physicochemical
properties may be utilized to determine the stoichiometry of the complexes formed
and numerical values of their stability constants. These include changes in solubility,
changes in chemical reactivity, changes in UV/Vis absorbance, changes in
fluorescence, NMR chemical shift, changes in drug retention (e.g., in liquid
chromatography), changes in pka values, potentiometric measurement, changes in
chemical stability and effects on drug permeability through artificial membranes.
Furthermore, since complexation will influence the physicochemical properties of the
aqueous complexation media, methods that monitor these media changes can be
applied to study the complexation.
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For example, measurements of conductivity changes, determination of
freezing point depression, viscosity measurements and calorimetric titration’s.
However, only few of these methods can be applied to obtain structural information
on drug/ cyclodextrin complexes.
1.7. Stability Studies:
(Lachmann L. and Herbert A. Libberman., 1991)
Stability of a drug can also be defined as the time from the date of
manufacture and packaging of the formulation until its chemical or biological activity
is not less than a predetermined level of labeled potency and its physical
characteristics have not changed appreciably or deleteriously. The environmental
factors, ingredients used and the nature of the container can affect stability.
Loss of potency usually occurs from a chemical change, the most common
reaction being hydrolysis, oxidation and reduction. Potency is determined by means
of assay procedure that differentiated between the intact drug and its degradation
product.
Accelerating the decomposing process and extrapolating the result to normal
storage conditions may make a prediction of the life of the product. Acceleration of
chemical decomposition is achieved by raising the temperature of the preparations.
Application of the principles of chemical kinetics to the results of accelerated storage
test carried out at three or more elevated temperatures enable prediction to be made of
the effective life of the preparation at normal temperature.
Plotting the appropriate function of concentration against time and obtaining a
linear relationship determine the order of the reaction for the decomposition process.
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The reaction velocity constant k for the decomposition at each of the elevated
temperature can be calculated from the slope of the line. The most satisfactory
method for expressing the influence of temperature on reaction velocity is the
quantitative relation proposed by Arrhenius.
–Ea/RT
Where, K = Ae
K = Specific rate of degradation.
R = Gas Constant (1.987 cals/deg/mol)
T = Absolute temperature.
A = Frequency factor.
Ea = Energy of activation.
The Arrhenius relationship is then employed to determine the ‘K’ value for
decomposition at room temperature. This is obtained from the linear plot of the
logarithm of ‘k’ value against reciprocal of absolute temperature, which is then
extrapolated to room temperature (25° C). The value of ‘K’ at 25° C may be then
substituted in the appropriate rate equation and an estimate obtained of the time
during which the product will maintain the required quality or potency (shelf- life).
The table below indicates maximum time and minimum time at which potency
must be at least 90 % of label claim at the temperature indicated in order to predict a
shelf- life of two years at room temperature.
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Table 1.2: Stability requirement for maintenance of shelf- life
Temperature° C
Maximum time
Minimum time
37° C
12 months
6.4 months.
45° C
8.3 months
2.9 months.
60° C
4.1 months
3 weeks.
85° C
6 weeks
2.5 days
If the assay is over 90 % of original potency at the minimum time (with
activation energy 20 K cals/mol) at the respective temperature, in all probability the
assays will be over 90 % after two years at room temperature. If the assay remain
over 90 % at the maximum time shown (with activation energy 10 K cals/mol) it is
certain that a potency of over 90 % will be maintained after two years at room
temperature.
1.8. Applications of Cyclodextrins:
(Higuchi T. and Connors K.A., 1965; Rao B.P et al., 2007; Suresh S et al., 2004)
™ β-Cyclodextrin Inclusion Complexes:
Bioavailability Enhancement: Drugs with poor bioavailability typically have low
water solubility and/or tend to be highly crystalline. As cyclodextrins is water soluble
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and form inclusion complexes with apolar molecules or functional groups in water
insoluble compounds.
The resulting complex hides most of the hydrophobic functionality in the interior
cavity of the cyclodextrin while the hydrophilic hydroxyl groups on its external
surface remain exposed to the environment. The net effect being a water-soluble
cyclodextrin-drug complex. In addition to improving solubility, cyclodextrins also
prevent crystallization of active ingredients by complexing individual drug molecules
so that they can no longer self-assemble into a crystal lattice.
Active Stabilization: For an active molecule to degrade upon exposure to radiation,
heat, oxygen or water, chemical reactions must take place. When a molecule is
constrained within the cyclodextrin cavity, it is difficult for reactants (water or
oxygen) to diffuse into the cavity and react with the protected guest. In the case of
thermal or radiation induced degradation, the active must undergo molecular
rearrangements. Again, due to the steric constraints on the guest molecule within the
cavity, it is difficult for the active to fragment upon exposure to heat or light or if it
does fragment, the fragments do not have the mobility needed to separate and react
before a simple recombination takes place.
Odour or Taste Masking: Through encapsulation within the cyclodextrin cavity,
molecules or specific functional groups that cause unpleasant tastes or odours are
hidden from the sensory receptors. The resulting formulations have no or little taste or
odour and are much more agreeable to the patient.
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Compatibility Improvement: Often one would like to combine multiple ingredients
or drug actives within a single formulation due to the potential for synergistic
benefits. However, different drugs are often incompatible with each other or another
inactive ingredient within a formulation. Encapsulating one of the incompatible
ingredients within a cyclodextrin molecule stabilizes the formulation by physically
separating the components in order to prevent chemical interaction.
Material Handling Benefits: Active ingredients that are oils/ liquids or are volatile
materials can be difficult to handle and formulate into stable solid dosage forms.
Encapsulating these types of substances in a cyclodextrin converts them to a solid
powder that has good flow properties and can be conveniently formulated into a tablet
by conventional production processes and equipment.
Irritation Reduction: Active ingredients that irritate the stomach, skin or eye can be
encapsulated within a cyclodextrin to reduce their irritancy. The formation of an
inclusion complex reduces the local concentration of free active ingredient below the
irritancy threshold. As the complex gradually disassociates, the active ingredient is
absorbed into the body for therapeutic benefits, but its local free concentration
remains below levels that might be irritating.
Oral Drug Delivery System: Rapid dissolving complexes with cyclodextrin have
also been formulated for buccal and sublingual administration in this type of drug
delivery system a rapid increase in the systemic drug concentration takes place along
with the avoidance of systemic and hepatic first pass metabolism such as enhanced
solubility of the ofloxacin drug.
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Industrial Applications: Cyclodextrin change the physical and chemical properties
of organic compounds. By this unique characteristic, cyclodextrins are used
extensively in pharmaceutical, food and other industries for the following purpose:
• Suppression of volatility of volatile compounds.
• Stabilization of labile components decomposed and denatured by oxidation, UV-
irradiation, hydration, heating, freezing and drying.
• Drying assistance of humid or liquid food extracts, seasoning and beverages.
• Reduction of bad taste and odour.
• Emulsification of oily material, solubilization water- in-soluble substances and
removal of non-volatile compounds from food.
Potential Applications of β-Cyclodextrin: Cyclodextrin iodine complexes (CDS) by
Nippon Chemical Co. Ltd., Japan are antibacterial deodorants, wrapping iodine in
cyclodextrin cavity. CDIs possess powerful antibacterial activity with broad spectrum
β-cyclodextrin iodine complexes are used in powder formulation.
LITERATURE
SURVEY
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2. LITERATURE SURVEY
2.1. Literature Review:
Recent advancements in Inclusion complexes:
1. Akbari B.V et al., (2011) had developed cyclodextrin complexes to increase
the solubility, and dissolution rate of Rosuvastatin Calcium (RST), a poorly
water- soluble 3-hydroxy3-methyl glut, aryl (CoA (HMG-CoA) Reductase
inhibitor through inclusion complexation with β–cyclodextrin (β-CD). The
inclusion complexes were prepared by three different methods viz. physical,
kneading, Co- evaporation and precipitation method. The inclusion complex
prepared with β–CD by kneading method exhibited greatest enhancement in
solubility and fastest dissolution (98.96 % RST release in 30 min) of RST.
2. Vavia P.R et al., (1999) focused on inclusion complexation of ketoprofen with
β– cyclodextrin (β-CD) and hydroxypropyl β–cyclodextrin (HP-β-CD). The solid
complexes were prepared by various methodologies such as physical mixture co-
precipitation, kneading and freeze drying. The drug and cyclodextrins were used
in molar ratio of 1:1. Freeze drying method was found to be the method of choice
for successful inclusion complexation of ketoprofen with β-CD and HP-β-CD.
3. Bushetti S.S et al., (2009) the work was to improve the solubility, dissolution
rate and antibacterial activity of drug by formulating its inclusion complexes with
β- cyclodextrin and hydroxyl propyl β-cyclodextrin by kneading and physical
mixture methods. Drug release profile was studied in 7.2 pH phosphate buffer.
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The results showed that an improvement in the antibacterial activity of the drug
in its inclusion complex with hydroxyl propyl β-cyclodextrin in 1:3 molar ratio.
4. Patil J.S. et al., (2010) studied about orally administered drugs completely absorb
only when they show fair solubility in gastric medium and such drugs shows good
bioavailability. Solid dispersion, solvent deposition, micronization are some vital
approaches routinely employed to enhance the solubility of poorly water soluble
drugs. Cyclodextrins, the unique cyclic carbohydrates are successfully utilized as
the potential complexing agents which form inclusion complex with insoluble drugs
of various techniques have been investigated to explain the methods for preparation
of inclusion complexes. In the present review, an attempt was made to discuss
various complexation techniques and tried to briefly highlight the potential
applications, technical and economical limitations associated with these approaches.
5. Ramink singh et al., (2010) A review conducted on CD, cyclodextrins are cyclic
oligosaccharides which have recently been recognized as useful pharmaceutical
excipients. The molecular structure of these glucose derivatives generates a
hydrophilic exterior surface and a non polar cavity interior. Such cyclodextrins can
interact with appropriate size drug molecules which lead to the formation of
inclusion complexes. The characterization of inclusion complexes was done with a
purpose to determine the interaction of drug molecules with cyclodextrins which
confirm the formation of inclusion complexes.
6. Swaroop S. et al., (2007) were formulated the inclusion complex of curcumin with
β-cyclodextrin (β-CD) has been characterized by absorption and fluorescence
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spectroscopy. From temperature-dependent fluorescence measurements, the
thermodynamic parameters ∆H and ∆S, for the complexation were estimated.
Kinetics of binding was studied by stopped flow technique, from which the binding
constant for inclusion complex was estimated. Further, influence of such inclusion
complex on change in superoxide radical scavenging property of curcumin was
examined using xanthine/xanthine oxidase assay.
7. Guowang Diao et al., (2006) A research work on CD, β-Cyclodextrin can react with
nitrobenzene to form an inclusion complex which is characterized by 'H NMR and
power X-ray diffractometry. The ratio of β-CD to NB in inclusion complex is
determined as 1:1. At 25° C, the dissociated constant, KD, of the inclusion complex
is measured as 6.5 x 10-3 M in neutral solution (pH=7.0), but in alkali (pH=13.5), KD
is 3.2 x 10-2 M which is much larger than that measured in neutral.
8. Shewale B.D et al., (2008) were examined the effect of pH and concentrations of
hydroxyl propyl β-cycloedextrin on the solubility of carvedilol as it shows pH-
dependent solubility. But inclusion in the cavity of hydroxyl propyl-β-cyclodextrin
might depend upon charge state of the molecule. So it can be concluded that
solubility of carvedilol, can be increased either by the addition of hydroxypropyl-β-
cyclodextrin or by adding pH lowering agents. But both these methods if are to be
used together, pH should be selected carefully.
9. Indap M.A et al., (2002) had suggested hydroxyl propyl beta cyclodextrin complex
was able to protect bone marrow cells from lethal effect of radiation. When the
cytotoxicities of quercetin and its complexes were compared on erythrocytes of rat
Inclusion Complexes of Aceclofenac Literature survey
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and rabbits, no significant differences were observed. The ability to selectively
target quercetin via its cyclodextrin inclusion complex against cancer growth could
improve the therapeutic effectiveness of cyclodextrin preparations as well as reduce
adverse side effects associated with quercetin. The new cyclodextrin inclusion
complex appears to have high potential for the treatment of leukemia’s and possibly
also for solid tumors.
10. Chowdary K.P.R et al., (2002) the complex formation of nifedipine with β-
cyclodextrin and hydroxyl propyl β-cyclodextrin was studied. The phase solubility
studies indicated the formation of a nifedipine-β-cyclodextrin (1:1 M) and
nifedipine-hydroxypropyl β-cyclodextrin (1:1 M) inclusion complexes with a
stability constant of121.9M-1 and 253.7M-1 respectively.
11. Chowdary K.P.R et al., (2006) tested the solubility and dissolution rate of celecoxib
were markedly enhanced by complex with β-cyclodextrin and hydroxyl propyl-β-
cyclodextrin. Celecoxib-hydroxypropyl-β-cyclodextrin (1:2) inclusive complex
gave a 36.57-fold increase in the dissolution rate of celecoxib. Addition of
polyvinylpyrrolidone results in higher complexation efficiency and markedly
enhanced solubilizing efficiency of β-cyclodextrin and hydroxypropyl-β-
cyclodextrin.
12. Jagtap Rajesh S. et al., (2009) enhanced the solubility and impart a controlled
release in a single formulation. Glipizide, an oral hypoglycemic agent which
belongs to Class II of BCS with relatively short elimination half life (2-4 hour) was
complexed with β-cyclodextrin. The dissolution study of kneading complex shows
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significant increase in the drug release from kneading complex than pure drug and
physical mixture. The formulation F3 having Similar dissolution profile like
marketed preparation (GLUCOTROL XL) which having 50 mg of PEO. Finally it
can be concluded from the study that molecular complex with β-CD play a vital to
obtain a uniform, controlled and complete drug release of a poorly soluble drug from
the swellable / erodible matrix tablet.
13. Sanoferjan A.M et al., (2000) had formulated the cyclodextrin complexation of
tenoxican was attempted to enhance the solubility features of the drug. The complex
prepared in 1:1 M ratio by various techniques was evaluated for its dissolution
profile, thermal stability and photostability. The complex prepared by neutralization
method was found to yield very reliable and best results over that of the common
solvent and kneading method.
14. Ramana M.V et al., (2008) showed that enhanced dissolution rate of rofecoxib in
both media from betacyclodextrin complex and the further enhancement of
dissolution was found in presence of ascorbic acid and citric acid and thus
betacyclodextrin enhances the solubility by creating the acidic environment around
the drug molecule, solubility of rofecoxib could be enhance further, which in turn
would enhance the absorption of rofecoxib and produced greater pharmacological
activity.
15. Deshmukh S.S et al., (2007) made an attempt to prepare and characterized inclusion
complexes of ziprasidone HCl with β-cyclodextrin and HP-β-cyclodextrin. The
phase solubility analysis indicated that the formation of 1:1 molar inclusion complex
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of a drug with β-cyclodextrin and HP-β-cyclodextrin. The inclusion complexes were
prepared co-precipitation, kneading and microwave method. The prepared
complexes were characterized using solubility study, DSC and XRD. The inclusion
complexes prepared with HP-β-cyclodextrin by microwave method exhibited
greatest enhancement in solubility and fastest dissolution of ziprasidone HCl.
16. Sarasija Suresh et al., (2006) made an attempt to enhance the solubility features o f
the drug. Carbamazepine β-cyclodextrin complex prepared by kneading method was
used to produce dispersible tablets. A 23 factorial design was employed to
investigate the effect of factors such as amount of binder, hardness and type of
disintegrant on the tablet disintegration time and dissolution rate. The three main
factors studied had a significant influence on both response parameters.
17. Sanjula Baboota et al., (2005) have prepared inclusion complexes of rofecoxib by
using dimethyl β-cyclodextrin. Complexes were prepared by physical, kneading and
spray drying methods. The release profile of the drug from complexes were studied
in pH 1.2 and pH 7.4 and it was found that the marketed preparations showed lesser
release characteristic as compared to the complex prepared by kneading method.
18. Aithal K.S et al., (2005) had prepared the complexes of the norfloxacin with β-CD
and enhanced UV absorption (a=3 times) and fluorescence emission (k=4 times) was
observed. The 1:1 M neutralization complex gave maximum values of both UV
absorption and fluorescence emission compared to physical mixture or kneading
complex.
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19. Dhaneshwar S.R et al., (2004) had prepared the methotrexate pro-drugs of α and γ -
cyclodextrin. The primary hydroxyl group of α and γ-cyclodextrin block the acidic
group and the hydrolysis of cyclodextrin conjugates in colon is confirmed by
hydrolysis kinetic studies in rat fecal material and the conjugate showed good
masking ulcerogenic potential of free drug.
20. Nagesh Bandi et al., (2004) had determined whether budesonide and indomethacin
HPβ-CD complexes could be formed using a supercritical fluid process. The process
involved the exposure of drug-HP-β-CD mixtures to supercritical carbon dioxide.
The ability of supercritical fluid process to form complexes was assessed by
determining drug dissolution, drug crystallinity and drug excipients interactions.
Thus budesonide and indomethacin HP-β-CD complexes with enhanced dissolution
can be formed using a single step organic solvent free SCF process.
21. Sanjula Baboota et al., (2005) had developed the inclusion complexes of
meloxicam with β-cyclodextrin by various methods like grinding, kneading, solid
dispersion and freeze-drying. The in-vitro dissolution rate of drug β-cyclodextrin
complex was faster compared to the drug alone.
22. Saha RK et al., (2002) had prepared inclusion complexes of nimesulide with β-
cyclodextrin by solvent evaporation method and solid dispersion of nimesulide and
ibuprofen by using various hydrophilic excipients (PEG-6000, sorbitol, PVPK-30,
MCC). Solid dispersion of nimesulide with PEG-6000 enhanced the solubility of
nimesulide more than 1000 %. The dispersion of ibuprofen in sorbitol showed
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maximum enhancement of solubility up to 75 %. The inclusion complexes of
nimesulide in β-cyclodextrin also increase the solubility by 663 %.
23. Dalmora M.E et al., (2001) evaluated the topical formulations of piroxicam by
determination of their in vitro release and in vivo anti- inflammatory. Piroxicam was
incorporated in ME (micro emulsion) and in the combined system β-cyclodextrin /
ME, respectively relative to the buffered piroxicam (42.2 %). These results
demonstrated that micro emulsion induced prolonged effects, providing inhibition of
the inflammation for 9 days after a single dose administration.
Literature Review on Selected drug Aceclofenac:
24. Thiyagarajan Ayyappan et al., (2009) had formulated the solid dispersion tablet
formulation of Aceclofenac, a novel non steroidal anti inflammatory drug mainly
used for rheumatoid arthritis osteoarthritis and ankylosing spondylitis. Aceclofenac
solid dispersion with crosspovidone showed maximum drug release and hence, the
tablet formulation containing Aceclofenac, crosspovidone and polyvinyl pyrolidone
K-30 solid dispersion, was prepared with a view to improve its water solubility. The
drug profile was studied in 2 % w/v sodium lauryl sulphate in Distilled water. F-III
gave far better dissolution than other laboratory developed formulations. The
formulation was found to be stable for 30 days at 40° C± RH 75 % as per ICH
guidelines, with insignificant change in the hardness, disintegration time, and in vitro
drug release pattern.
25. Kamal Dua et al., (2011) had developed and compared the stability of the β-CD
dimer-Aceclofenac complex with β-CD monomer-Aceclofenac. Such molecular-
Inclusion Complexes of Aceclofenac Literature survey
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modeling studies can be employed as an additional tool to support the formation of
stable molecular inclusion complexation of any water insoluble drug complexed with
cyclodextrins.
26. Dahiya S. et al., (2006) was prepared and characterized the binary systems of
Acelofenac (AC) with hydroxypropyl beta-cyclodextrin (HP-β-CD) in equimolar
ratio. Solid binary systems of Aceclofenac with HP-β-CD were prepared using co
grinding, kneading, and co evaporating methods, and a physical mixture was
prepared for comparison. All the binary systems showed superior dissolution and
lower dose: solubility ratio (D: S ratio) as compared to pure AC. It was suggested
that complexation of Aceclofenac with HP-β-CD may be used as an approach to
change the drug from Biopharmaceutics Classification System BCS Class II to BCS
Class I without changing its intrinsic permeability.
27. Gajendran P. et al., (2010) tested the phase solubility profile of Aceclofenac with
beta-cyclodextrin (β-CD) prepared by kneading method and those were
characterized by X-ray diffraction and FTIR spectra. The complexes were prepared
as tablets with and without sodium starch glycolate (2 %) (SSG) as super
disintegrants. Precompression parameters like tapped density, Carr’s index, bulk
density and angle of repose were determined. The order of faster dissolution rate
observed in various ratios with SSG was 1:2 complex > 1:1 complex >2:1 complex >
pure drug, in acidic medium contains 1 % SLS. Another batch was also prepared in
the same ratio with β-CD, this batch have higher solubility of Aceclofenac when 2 %
SSG added; the release was 30 min faster in 1:2 pH medium containing 1 % sodium
lauryl sulphate (SLS) from the above result we conclude the inclusion complex of
Inclusion Complexes of Aceclofenac Literature survey
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drugs and β-CD ratio 1:2 with SSG as superdisintegrate could be used as an effective
technique for immediate release of Aceclofenac.
28. Kulkarni N.S et al., (2010) carried out research work by developing the inclusion
complexation of Aceclofenac with beta-cyclodextrin by grinding, microwave and
spray drying techniques. The samples were subjected to in vitro dissolution studies,
fourier transform infra-red spectroscopy, DSC, NMR and X-ray diffraction studies.
They were reported in vitro dissolution of Aceclofenac-hydroxy propyl-beta-
cyclodextrin complex was faster as compared to the Aceclofenac-beta-cyclodextrin
complex and Aceclofenac alone.
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2.2. DRUG PROFILE
(IP., 2007; BP., 2009; Kabir., et al., 2009; Merck index., 2006)
ACECLOFENAC:
Aceclofenac is a potent non-steroidal anti- inflammatory drug. Due to its
preferential cox-2 blockade it has better safety than conventional NSAIDs with respect to
adverse effects on gastro intestinal and cardiovascular system.
IUPAC Name : 2-[(2, 6-Dichlorophenylamino) phenyl] acetoxy acetic acid.
Description : A white to almost white crystalline powder.
Structure :
Fig. 2.1: Structure of Aceclofenac
Molecular formula : C16H13Cl2NO4
Molecular weight : 354.2
Category : Non-steroidal anti inflammatory drug.
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Solubility : It is practically insoluble in water; soluble in alcohol and
methyl alcohol; It is freely soluble in acetone and dimethyl
formamide.
Melting point : 149 - 153° C
Pharmacology:
1. Aceclofenac directly blocks PGE2 secretion at the sight of inflammation by inhibiting
IL- Beta TNF in the inflammatory cells. (Intracellular action)
2. Aceclofenac stimulates the synthesis of the extra cellular matrix of the human articular
cartilages.
3. Inhibits neutrophil adhesion and accumulation at the inflammatory sites in the early
phase and thus blocks the pro- inflammatory action of neutrophil.
4. Aceclofenac has higher COX2 specificity than diclofenac sodium.
. Fig. 2.2: Pathway describing general anti- inflammatory action of Aceclofenac
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Pharmacokinetics:
Aceclofenac is rapidly and completely absorbed after oral administration, peak
plasma concentration is reached 1 to 3 hours after an oral dose. The drug is highly protein
bound. The presence of food does not alter the extent of absorption of Aceclofenac but
the absorption rate is reduced. It is metabolized to a major metabolite
4-hydroxyAceclofenac and to a number of other metabolites including 5–Hydroxy
Aceclofenac, 4-Hyrodxydiclofenac, Diclofenac and 5-Hydroxydiclofenac. Renal
excretion is the main route of elimination of Aceclofenac with 70 to 80 % of an
administered dose found in the urine, mainly as the glucuronides of Aceclofenac and its
metabolites of each dose of Aceclofenac, 20 % is excreted in the faeces. The plasma
elimination half life of the drug is approximately 4 hours.
Drug interactions:
Aceclofenac may increase plasma concentrations of lithium, digoxin and
methotrexate, increase the activity of anticoagulant, inhibits the activity of diuretics,
enhance cyclosporine nephrotoxicity and precipitate convulsions when co-administered with
quinolone antibiotics. Furthermore, hypo or hyperglycemia may result from the
concomitant administration of Aceclofenac and antidiabetic drugs, although this is rare.
The co-administration of Aceclofenac with other NSAIDs results in increased frequency
of adverse event.
Adverse drug interactions:
Aceclofenac is well tolerated with, most adverse events being minor and
reversible and affecting mainly the G.I system. Most common events include dyspepsia
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(7.5 %), abdominal pain (6.2 %), nausea (1.5 %), diarrohoea (1.5 %), flatulence (0.8 %),
gastritis (0.6 %), constipation (0.5 %), vomiting (0.5 %), and pancreatitis (0.1 %). Other
adverse effects which is not common such as dizziness (1 %), vertigo (0.3 %), and rare
cases of paraesthesia and tremor.
Dosage and administration : The usual dose of Aceclofenac is 100 mg given
twice daily by mouth, one tablet in the morning and
one in the evening.
Storage : In an air tight container, protected from light.
Uses : Aceclofenac is used in the treatment of
Osteoarthritis, rheumatoid arthritis, ankylosing
spondylitis, dental pain, post operative pain,
dysmenorrhoea, acute lumbago, musculoskeletal
trauma and gonalgia (knee pain).
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2.3. β-CYCLODEXTRIN PROFILE
(Raymond C.Rowe., 2003)
β-CYCLODEXTRIN:
Synonyms : Beta-cycloamylose; betadex; beta-dextrin; cyclohepta
amylase; Cycloheptaglucan: cyclomaltoheptose; kleptose.
Functional category : Solubilizing agent and stabilizing agent.
Empirical Formula : C42 H70O35
Molecular Weight : 1135.
Fig.2.3: Structure of β-Cyclodextrin
Description : White, practically odorless, amorphous powder, having a
slightly sweet taste.
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Solubility : Soluble in 1 in 200 part of propylene glycol, 1 in 50
of water at 20° C, 1 in 20 at 50° C.
Stability and storage conditions: Stable in the solid state if protected from high
humidity, should be stored in a tightly sealed
container in cool, dry place.
Safety : Used in oral pharmaceutical formulations.
Handling Precaution : It should be handled in a well-ventilated
environment efforts should be made to limit the
generation of dust, which can be explosive.
AIM
AND
OBJECTIVE
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3. AIM AND OBJECTIVE
The poor aqueous solubility and dissolution of relatively insoluble drugs has
been a major problem for a long time in the formulation of oral dosage form. So in
the recent years much attention has been focused on the problem of drug
bioavailability. The dissolution rate of a drug from its dosage form now considered as
an important parameter in bioavailability, dissolution is the rate limiting step in the
absorption of the drug from solid dosage forms, especially when the drug is poorly
water soluble. Among the various approaches to improve the dissolution of the drugs,
the preparation of cyclodextrin inclusion complexes has often proven to be highly
successful.
It has short biological half-life (4 to 4.3 hours), and the usual oral dosage
regimen is 100 mg taken 2 times a day. The basic goal of therapy is to achieve a
steady state blood or tissue level that is therapeutically effective and non-toxic for the
treatment period of time.
Aceclofenac is Nonsteroidal anti-inflammatory drug (NSAID). It is phenyl
acetic acid derivative showing effective anti-inflammatory, analgesic and anti-pyretic
properties mainly used in osteoarthritis and rheumatoid arthritis.
Aceclofenac is practically insoluble in water. Its aqueous solubility is reported
as 0.53 mg/ml. The aqueous solubility of drug is improved by various techniques like
solid dispersions, micronization, solvent deposition, use of surfactants and inclusion
complexation etc.
Inclusion Complexes of Aceclofenac Aim and Objective
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In the present work, was aimed to prepare and characterize the inclusion
complexes of Aceclofenac with cyclodextrins in different molar ratios by different
methods such as physical, kneading and common solvent method.
Preparation of inclusion complex using cyclodextrin for its ready water-
solubility, significant potential for solubilization and good safety profile.
The potential applications of Aceclofenac inclusion complexes:
¾ May minimize the frequent dosing intervals.
¾ Prolong the pharmacological effect.
¾ May help to improve patient compliance by reducing the gastrointestinal side
effects like peptic ulcer etc.,
PLAN OF WORK
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4. PLAN OF WORK
LITERATURE SURVEY
SELECTION OF DRUG AND β-CYCLODEXTRIN
PROCUREMENT OF DRUG AND β-CYCLODEXTRIN
EXPERIMENTAL WORK
Preformulation Study
Identification of Drug
€ Organoleptic Properties
€ Determination of Melting Point
€ Solubility Study
€ Loss on Drying
€ FTIR
€ UV Spectrophotometric Study in Methanol
€ UV Spectrophotometric Study in Distilled Water
€ Assay of Aceclofenac
Formulation of complexes with β-Cyclodextrin
€ Kneading method
€ Common Solvent method
€ Physical mixtures
Evaluation of inclusion Complexes
Phase Solubility Studies
€ UV Spectrophotometric Study of Prepared Inclusion Complexes
€ Fourier Transform Infrared (FTIR) Spectroscopy
€ Differential Scanning Calorimetry (DSC) Analysis
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€ X-ray Diffraction Studies
€ Drug Content Estimation
€ In vitro Dissolution Studies
€ Stability Studies
RESULTS AND DISCUSSION
SUMMARY AND CONCLUSION
FUTURE PROSPECTUS
BIBLIOGRAPHY
MATERIALS
AND
EQUIPMENTS
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5. MATERIALS AND EQUIPMENTS
5.1. Materials Used:
Table 5.1: List of raw materials with source
S. No Name of Ingredients Name of supplier
1 Aceclofenac Tristar Pharmaceuticals, Puduchery.
2 β - Cyclo dextrin Alkem laboratories pvt ltd, Mumbai.
3 25 % ammonia Qualigens fine chemicals, Mumbai.
4 Calcium chloride Qualigens fine chemicals, Mumbai.
5 Methanol Qualigens fine chemicals, Mumbai.
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5.2. Equipments Used:
Table.5.2: List of equipments with model/make
S. No Equipment Model/ Make
1 Electronic balance Shimadzu BL-220H.
2 Hot air oven Chemi Equipments, Bombay.
3 USP tablet dissolution apparatus
Type I
Veego scientific VDA-8DR.
4
UV spectrophotometer
Shimadzu-1700 Pharmaspec.
5 FTIR spectrophotometer JASCO FT/IR-5300
6
Differential scanning calorimeter
Shimadzu DSC 60, Japan.
7
Rotary Shaker
Remi Electrical
8
pH Meter
Elico Li-120
9
Melting Point Apparatus
Guna Enterprises, Chennai.
10
Standard sieve (40 #)
Jayant scientific, IND.
11
X-ray Diffractometer
X-ray Diffractometer PW-1710
PREFORMULATION STUDIES
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6. PREFORMULATION STUDY
6.1. Identification of Drug:
The preliminary studies were carried out by testing of different physical and
chemical properties of drug as follows.
6.1.1. Organoleptic properties: (Lachman L. et al., 1991)
The Organoleptic properties like physical state, colour, odour etc., of the drug
was reported with help of the descriptive terminology. It helps to identify the drug.
6.1.2. Melting point:
(IP., 2007)
It is the easy way to identify the drug. The melting point of Aceclofenac was
tested by use of a laboratory melting point apparatus with a procedure given in the
Indian Pharmacopeia 2007.
6.1.3. Solubility study:
(IP., 2007)
The solubility of Aceclofenac was determined by micropipette method in
various solvents in order to meet the official standards.
6.1.4. Loss on drying:
(IP., 2007)
Loss on drying is the loss of weight expressed as percentage w/w resulting
from water and volatile matter of any kind that can be driven off under specified
condition. The test can be carried out on the well mixed sample of the substance.
Initial weight of substance – Final weight of substance Loss on drying = -------------------------------------------------------------------- x 100
Initial weight of substance
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6.1.5. FTIR spectroscopy:
The infrared spectrum was generally used as an identification parameter to
know the chemical structure of drugs. For the FTIR spectrum of Aceclofenac, FTIR
spectrophotometer was used.
A small quantity of sample was mixed with sufficient potassium bromide and
compressed into a pellet by applying a 10 tons pressure with help of a hand operated
press. This pellet was kept in a sample holder and scanned from 4000 to 400 cm-1.
6.1.6. UV spectrometric study in Methanol:
¾ Determination of λmax:
Weighed amount of Aceclofenac was dissolved in Methanol to obtain a
1000 mcg/ml solution. This solution was subjected to scanning between 200-
400 nm and absorption maximum was determined. The effect of dilution on
absorption maxima was studied by diluting the above solution to 15 mcg /ml
and scanned from 200-400 nm. The wave length of maximum absorbance was
found to be 275 nm.
¾ Preparation of standard curve of Aceclofenac in Methanol:
(IP., 2007)
The standard curve was prepared by dissolving 50 mg of Aceclofenac
50 ml of Methanol. In the stock solution 1 ml was withdrawn and diluted to
25 ml of Methanol. It was further diluted with Methanol to get the solution in
the concentration range of 4-20 μg/ml. The absorbance values were
determined at 275 nm. A graph of absorbance Vs concentration was plotted.
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6.1.7. UV Spectrometric study in Distilled Water:
¾ Preparation of standard curve of Aceclofenac in Distilled Water:
(Rohit Shah et al., 2008)
The standard curve was prepared by dissolving 50 mg of Aceclofenac
in 50 ml of Methanol. In the stock solution 1 ml was withdrawn and diluted to 25 ml
of Methanol. It was further diluted with Distilled Water to get the solution in the
concentration range of 4-20 μg/ml. The absorbance values were determined at 274
nm. A graph of absorbance Vs concentration was plotted.
6.1.8. Assay of Aceclofenac:
(IP., 2007)
Accurately weighed the powder equivalent to 100 mg of Aceclofenac was
transferred to 100 ml volumetric flask and the volume was made up to the mark with
Methanol. The mixture was filtered through whatmann filter paper No.41 and then 5
ml of filtrate was transferred to 50 ml volumetric flask and made up to the mark with
Methanol.1 ml aliquots of the sample were transferred and diluted to 10 ml with
Methanol to obtain strength as 10 µg/ml and then the absorbance was measured at 275
nm against Methanol as blank. The percentage purity of the drug was calculated by
using calibration graph method.
FORMULATION
OF
INCLUSION
COMPLEXES
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7. FORMULATION OF INCLUSION COMPLEXES
Table 7.1: Composition of inclusion complexes of Aceclofenac
Method
Formulation
Code
Drug to
carrier
Drug to
carrier
ratio
Drug
(g)
β-Cyclodextrin
(g)
Kneading
method
F1 ACE : β-CD 1:1 1 1
F2 ACE : β-CD 1:2 1 2
F3 ACE : β-CD 1:3 1 3
Common
solvent
method
F4 ACE : β-CD 1:1 1 1
F5 ACE : β-CD 1:2 1 2
F6 ACE : β-CD 1:3 1 3
Physical
method
F7 ACE : β-CD 1:1 1 1
F8 ACE : β-CD 1:2 1 2
F9 ACE : β-CD 1:3 1 3
Where,
ACE - Aceclofenac
β-CD - Beta-Cyclodextrin
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7.1. Methods of Preparation of Inclusion Complexes:
(Sanoferjan A.M., 2000; Chowdary K.P.R., 2000)
Kneading method:
β-Cyclodextrin was taken in a glass mortar and little water was added and
mixed to obtain a homogenous paste. Aceclofenac was then added slowly while
grinding. The mixture was ground for 1h during this process appropriate quantity of
water was added to maintain suitable consistency. The paste was dried in an oven at
40º C for 48 hr. The dried complex was taken for study.
Common solvent method:
Aceclofenac and β-Cyclodextrin were dissolved in 25 % ammonia and the
solvent was allowed to evaporate overnight at room temperature. The complex so
prepared was pulverized and sifted through sieve no.80.
Physical mixture:
Previously weighted drug and β- cyclodextrin mixture was blended in glass
mortar for about an hour and passed through sieve no.85 to get physical mixture and
stored in desiccator over fused calcium chloride.
EVALUATION OF
INCLUSION COMPLEXES
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8. EVALUATION OF INCLUSION COMPLEXES
8.1 Phase Solubility Studies:
(Chowdary K.P.R., 2006 and Babu R.J., 2004)
Phase solubility studies for Aceclofenac (ACE) complexes were performed to
determine how the complexes of cyclodextrin affect the solubility of the Aceclofenac.
These studies also determine the stoichiometry of drug:cyclodextrin complexes and
numerical values of their stability constants.
€ Phase solubility of Aceclofenac (ACE) with beta-cyclodextrin (β-CD)
Procedure:
For phase solubility studies of ACE, an excess of drug (200 mg) was added to
20 ml portions of Distilled water, each containing variable amount of β-cyclodextrin
-3
such as 0, 1, 3, 6, 9, 12, and 15 x 10
moles/liter. All the above solutions with variable
amount of β-cyclodextrins were shaken for 72 hours. After shaking, the solutions were
filtered and their absorbance was noted at 275 nm. The solubility of the ACE in every
β-cyclodextrin solution was calculated and phase solubility diagram was drawn
between the solubility of ACE and different concentrations of β-cyclodextrin.
The stability constant of ACE β-cyclodextrin complex was calculated using
Higuchi and Connor’s equation.
K (1:1) = Slope
S0 (1-Slope)
S = Intrinsic solubility of ACE in aqueous complexation media (Distilled water) “slope” 0
was calculated from phase solubility diagram.
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8.2. UV Spectrometric Study of Prepared Inclusion Complexes:
(Rohit shah et al., 2008)
Weighed accurately the quantity of inclusion complex equivalent to 50 mg was
transferred to 50 ml volumetric flask and volume was made up to the mark with
Methanol. The mixture was filtered through whatmann filter paper No.41 and then 5
ml of filtrate was transferred to 50 ml volumetric flask and made up to the mark with
Methanol. From the resulting solution 1ml was taken and diluted with 10 ml of
Methanol to get a final concentration of 10 µg/ml. The sample solutions were scanned
at 200-400 nm.
8.3. Fourier Transform Infrared (FTIR) Spectroscopy:
(Gajendran P. et al., 2010)
Infrared spectroscopy is one of the most powerful analytical techniques that
offer the possibility of chemical identification. The IR spectra of Aceclofenac, β-
cyclodextrin and inclusion complex were obtained by KBr pellet method by JASCO
FT/IR-5300 spectrometer.
8.4. Differential Scanning Calorimetry (DSC) Analysis:
(Gajendran P. et al., 2010)
The thermal behavior of Aceclofenac, β-cyclodextrin and inclusion complex
was studied using differential scanning calorimetry in order to confirm the formation
of the solid complex. When guest molecule are incorporated in the cyclodextrin cavity
or in the crystal lattice, their melting, boiling and sublimation points are usually shifted
to a different temperature or disappear within the temperature range, where the
cyclodextrin lattice is decomposed.
Inclusion Complexes of Aceclofenac Evaluation of Inclusion Complexes
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 61
8.5. X-ray Diffraction Studies:
(Srinivas Mutalik et al., 2008)
The X-ray diffraction patterns of Aceclofenac, β-cyclodextrin and inclusion
complex were reordered using Philips X-ray diffractometer (Model: PW 1710) with
copper target. The conditions were: voltage -30kV; current -30mA; scanning speed -
1°/min; temperature of acquisition; room temperature; detector: scintillation counter
detector; sample holder: non-rotating holder.
8.6. Drug Content Estimation:
(IP., 2007)
Accurately weighed the quantity of inclusion complex equivalent to 100 mg
of drug was transferred to 100 ml volumetric flask and the volume was made up to the
mark with Methanol. The mixture was filtered through whatmann filter paper No.41
and then 5 ml of filtrate was transferred to 50 ml volumetric flask and made up to the
mark with Methanol.1 ml aliquots of the sample were transferred and diluted to 10 ml
with Methanol to obtain strength as 10 µg/ml and then the absorbance was measured at
275 nm against Methanol as blank.
8.7. In vitro Dissolution studies:
(IP., 2007)
In vitro dissolution of Aceclofenac inclusion complex was studied in USP
XXIV dissolution apparatus employing an apparatus I, 900 ml of Distilled water was
used as dissolution medium. The speed of rotation was set at 50 rpm. The
temperature of dissolution media was previously warmed to 37±0.5° C and was
maintained throughout the experiment. 1 ml of sample of dissolution medium were
withdrawn at known intervals of time, filtered the solution and analyzed for the drug
release by measuring the absorbance at 274 nm after suitable dilution with Distilled
Inclusion Complexes of Aceclofenac Evaluation of Inclusion Complexes
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 62
Water. Then the volume withdrawn at each time interval was replaced with the fresh
Distilled Water. The percentage amount of Aceclofenac released was calculated and
plotted against time.
Table 8.1: Parameters were used for the dissolution study
Apparatus
USP Dissolution apparatus
(Type I)
Dissolution medium
Distilled Water
Temperature 37 + 0.5° C
Volume 900 ml
Speed 50 rpm
Sample withdrawn 1 ml
Running Time 60 min
8.8 Stability Studies:
(Sanoferjan AM et al., 2000)
The formulation F2 was packed in dessicator, which were tightly plugged with
cotton and capped. They were then stored at 25°
C at 60 % and 40° C at 75 % RH for a
period of three months and the samples were withdrawn at every month for the
estimation of drug content and in vitro dissolution studies.
RESULTS
AND
DISCUSSION
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 63
9. RESULTS AND DISCUSSION
9.1. Identification of Drug:
9.1.1. Organoleptic Properties:
Colour : White or almost white powder
Odour : Odourless
Appearance : White crystalline powder
9.1.2. Melting Point:
Melting point of Aceclofenac was found to be 150.3±21° C. The official
melting point range for Aceclofenac is between 149-153° C. Hence, results were
complied the limits specified in official Book.
9.1.3. Solubility Study:
Table 9.1: Solubility of Aceclofenac in various solvents
S. No.
Solvents
Extent of Solubility
Inference
1 Distilled Water 10 mg in more than 10 ml Insoluble
2 Ethanol 10 mg in 160 µl Soluble
3 Methanol 10 mg in 200 µl Soluble
4 0.1N HCl 10 mg in more than 10 ml Insoluble
5 0.1N NaOH 10 mg in more than 10 ml Insoluble
6 Acetone 10 mg in 70 µl Freely soluble
7 pH buffer 7.4 10 mg in more than 10 ml Insoluble
8 Chloroform 10 mg in 400 µl Sparingly soluble
The results were complied with the official book.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 64
9.1.4. Loss on Drying:
The percentage loss on drying after three hours was found to be 0.25 %.
Table 9.2: Percentage loss on drying of Aceclofenac
S. No.
Percentage LOD
Average
percentage LOD
1 0.25
0.25 ± 0.01 2 0.26
3 0.24
The sample passes the test for loss on drying as per limit specified in IP 2007
(NMT 1 %).
9.1.5. FTIR spectroscopy:
Fig 9.1: FTIR spectrum of Aceclofenac
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 65
Table 9.3: Characteristic frequencies in FTIR spectrum of Aceclofenac
S. No Wave number(cm-1
) Inference
1 3316.41 N-H stretching
2 3110.41 C-H stretch ing
3 1626.63 C=O stretch ing
4 1386.20 Aromat ic C-H stretching
The drug was confirmed as Aceclofenac with results obtained from FTIR
spectrum analysis.
9.1.6. UV spectrophotometric study:
¾ The absorption maximum for Aceclofenac in Methanol was found to be 275
nm
Fig. 9.2: λmax of Aceclofenac in Methanol
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 66
¾ The absorption maximum for Aceclofenac in Distilled Water was found to be
274 nm
Fig. 9.3: λmax of Aceclofenac in Distilled Water
9.1.7. Calibration curve of Aceclofenac in Methanol:
UV absorption spectrum of Aceclofenac in Methanol shows λ max at
275 nm. Absorbance obtained for various concentrations of Aceclofenac are given in
Table 9.4. The graph of absorbance vs concentration for Aceclofenac was found to be
linear in the concentration range of 4 – 20 μg /ml.
Table 9.4: Data of concentration and absorbance for Aceclofenac in Methanol
S. No. Concentration
(µg/ml)
Absorbance(nm)
1 0 0.000
2 4 0.110
3 8 0.209
4 12 0.327
5 16 0.414
6 20 0.484
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 67
Fig. 9.4: Standard graph of Aceclofenac in Methanol
Table 9.5: Data for calibration curve parameter of Methanol
S. No. Parameters Values
1 Correlation coefficient (r) 0.999
2 Slope 0.008
3 Intercept 0.034
9.1.8. Calibration curve of Aceclofenac in Distilled Water:
UV absorption spectrum of Aceclofenac in Distilled Water shows λ
max at 274 nm. Absorbances obtained from various concentrations of Aceclofenac in
Distilled Water are given in Table 9.6. The graph of absorbance vs concentration for
Aceclofenac was found to be linear in the concentration range of 4 – 20 μg/ml.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 68
Table 9.6: Data of concentration and absorbance for Aceclofenac in Distilled Water
S. No. Concentration (µg/ml) Absorbance (nm)
1 0 0.000
2 4 0.051
3 8 0.112
4 12 0.152
5 16 0.195
6 20 0.241
Fig. 9.5: Standard graph of Aceclofenac in Distilled Water
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 69
Table 9.7: Data for calibration curve Parameter of Distilled Water
S. No. Parameters Values
1 Correlation coefficient (r) 0.998
2 Slope (m) 0.014
3 Intercept(c) 0.064
9.1.9. Assay of Aceclofenac:
The percentage purity of drug was calculated by using calibration graph
method (least square method).
Table 9.8: Assay of Aceclofenac
S. No. Percentage purity (%) Avg. percentage purity (%)
1 98.32
99.69 ± 1.2095 2 100.16
3 100.60
*All the values are expressed as mean ± SD, n = 3.
The percentage purity of raw material Aceclofenac was found to be 99.69 %.
Hence, the sample declared as pure.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 70
9.2. Phase Solubility Studies: Table 9.9: Phase solubility studies of Aceclofenac - β-cyclodextrin complexes
Concentration of β-CD (mM)
Concentrations of Aceclofenac*
(mM)
0 0.651 ± 0.026
1 0.675 ± 0.045
3 0.701 ± 0.062
6 0.784 ± 0.027
9 0.869 ± 0.035
12 0.948 ± 0.042
15 1.056 ± 0.039
*All the values are expressed as mean ± SD, n=3.
Fig. 9.6: Phase solubility studies of Aceclofenac - β-cyclodextrin complexes
r2
- value = 0.991
B- value (slope) = 0.026
A- value (intercept) = 0.636
Stability constant – K (1:1) = Slope
S0 (1 – slope)
-3
= 0.026 /0.636×10
= 42.003.
(1-0.026)
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 71
The phase solubility revealed a linear relationship between aqueous drug
solubility with increase in β-cyclodextrin (r2= 0.991). The phase solubility results
were shown in Table 9.9 and Fig. 9.6.
9.3. UV Spectrometric Study of Prepared Complexes:
Fig. 9.7: λmax of prepared complexes
The λmax of Aceclofenac in Methanol shows 275 nm and prepared complex
(formulation F2) shows 276 nm. This slight change is due to formation of inclusion
complex.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 72
9.4. Fourier Transform Infrared (FTIR) Spectroscopy:
Fig. 9.8: FTIR spectrum of Aceclofenac
Fig. 9.9: FTIR spectrum of β-cyclodextrin
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 73
Fig.9.10: FTIR spectrum of formulation F2
Table 9.10: Interpretation of FTIR spectrum of Aceclofenac
S. No.
Wave
number
(cm-1
)
Functional
group
Peak observed (Yes/No)
Range
(cm-1
)
Drug Drug+ β-
CD
1
3316.41 N-H
stretching
3600-3300
Yes
Yes
2
3110.41 C-H
stretching Above
3000
Yes
Yes
3
1626.63 C=O
stretching
1760-1615
Yes
Yes
4
1386.20 Aromatic C-
H stretch ing
1600-1300
Yes
Yes
From the above Fig. 9.8 to 9.10, it can be seen that, the major functional group
peaks like 3316.41, 3110.41, 1626.63 and 1386.20 cm-1 showing the functional group
N-H stretching, C-H stretching, C=O stretching and aromatic C-H stretching were
observed in spectra of drug with β-cyclodextrin complex remains unchanged as
compared with spectra of Aceclofenac.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 74
So, from the above IR spectra can be observed that there is no interaction
between Aceclofenac and β-cyclodextrin.
9.5. Differential Scanning Calorimetry (DSC) Analysis:
Fig. 9.11: DSC thermogram of Aceclofenac
Fig. 9.12: DSC thermogram of β-cyclodextrin
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 75
Fig. 9.13 DSC thermogram of formulation F2
Table 9.11: Data for DSC thermogram parameters
S.No.
DSC
thermogram
sample
Onset
temperature
(° C)
Peak
temperature
(° C)
Endset
Temperature
(° C)
1 Aceclofenac 152.51 153.85 155.36
2
Aceclofenac
+ β-CD
151.22
152.24
153.47
The thermal behavior of Cyclodextrin inclusion complex was studied using
DSC in order to confirm the formation of inclusion complexes. When the molecules
are incorporated in cyclodextrin cavity or in the crystal lattice, the boiling point,
melting point and sublimation point are usually shifted to different temperature
otherwise disappear within the temperature range where the cyclodextrin lattice is
decomposed.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 76
The DSC of pure Aceclofenac, β-Cyclodextrin and formulation F2 complexes
are shown in Fig.9.11 – 9.13. The results of the study indicate that there are no major
changes between the drug and drug carrier with reference to the melting point of
Aceclofenac and β-Cyclodextrin.
9.6. X-ray Diffraction Studies:
Fig. 9.14: X-ray diffraction of pure Aceclofenac
Fig. 9.15: X-ray diffraction of pure β-cyclodextrin
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 77
Fig. 9.16: X-ray Diffraction of formulation F2
Powder X-ray diffraction study may be used to detect inclusion complexation
in the solid state. The diffraction pattern of newly formed substances clearly differs
from that of uncomplexed cyclodextrin. This difference of diffraction pattern
indicates the complex formation.
The X-ray diffraction pattern of pure drug shows the peaks that are sharp
intense signifying its crystalline nature. Formulation F2 consists of drug and β-
cyclodextrin in the ratio 1:2 prepared by kneading technique there is absence of
intense peaks of Aceclofenac, signifying amorphous nature of complex. The results of
the XRD pattern are shown in the Fig. 9.14-9.16.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 78
9.7. Drug Content Estimation:
Table 9.12: Drug content estimation of Aceclofenac inclusion complexes
Formulation Code
Percent drug content*±SD
F1
98.02±0.13
F2
99.51±0.61
F3
97.23±0.21
F4
96.21±0.58
F5
97.28±0.28
F6
95.38±0.31
F7
98.52±0.84
F8
99.01±0.21
F9
97.23±0.21
*All the values are expressed as mean ± SD, n=3.
Drug content was found to be uniform among all formulations and ranged
from 97.23 to 99.51 %. These results showed that all formulations having uniform
percentage drug content as per limits given in Indian Pharmacopoeia.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 79
Cu
mu
lati
ve %
dru
g re
leas
e
9.8. In vitro Dissolution Studies:
Table.9.13: Dissolution profile of formulation F1
S. No.
Time (min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
8.25±0.18
8.25
4.79
0.50
3
10
20.48±1.22
20.48
9.49
0.99
4
15
33.86±1.61
33.86
13.88
1.43
5
30
57.26±0.33
57.26
18.02
1.90
6
45
75.09±1.63
75.09
21.86
2.28
7
60
83.37±1.81
83.37
25.72
2.91
*All the values are expressed as mean ± SD, n=3.
100 90 80 70 60 50 40 30 20 10
0
F1 0 5 10 15 30 45 60
Time in minutes
Fig.9.17: Dissolution profile of formulation F1
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 80
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.14: Dissolution profile of formulation F2
S. No.
Time (min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
26.55±0.80
26.55
12.07
2.50
3
10
43.33±1.05
43.33
23.06
4.75
4
15
55.55±1.92
55.55
32.14
6.49
5
30
85.18±1.20
85.18
51.82
12.00
6
45
93.99±0.29
93.99
64.75
14.28
7
60
97.54±0.89
98.54
73.15
16.93
*All the values are expressed as mean ± SD, n=3.
100 90 80 70 60 50 40 30 20 10
0
F2 0 5 10 15 30 45 60
Time in minutes
Fig. 9.18: Dissolution profile of formulation F2
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 81
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.15: Dissolution profile of formulation F3
S. No.
Time
(min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
8.27±1.21
8.27
3.59
2.50
3
10
25.46±0.67
25.46
8.92
5.82
4
15
32.83±0.30
32.83
15.17
8.31
5
30
52.28±0.22
52.28
29.56
13.53
6
45
65.89±0.89
65.89
39.77
18.11
7
60
75.09±1.49
75.09
47.71
22.56
*All the values are expressed as mean ± SD, n=3.
100 90 80 70 60 50 40 30 20 10
0
F3 0 5 10 15 30 45 60
Time in minutes
Fig. 9.19: Dissolution profile of formulation F3
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 82
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.16: Dissolution profile of formulation F4
S. No.
Time (min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
11.89±1.33
11.89
5.71
2.50
3
10
24.68±0.89
24.68
11.04
4.82
4
15
32.37±1.25
32.37
16.59
7.69
5
30
51.09±1.53
51.09
29.56
12.62
6
45
61.28±1.40
61.28
38.83
17.58
7
60
72.85±1.97
72.85
45.76
20.43
*All the values are expressed as mean ± SD, n=3.
100 90 80 70 60 50 40 30 20 10
0
F4 0 5 10 15 30 45 60
Time in minutes
Fig.9.20: Dissolution profile of formulation F4
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 83
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.17: Dissolution profile of formulation F5
S. No.
Time (min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
10.45±1.17
10.45
5.00
2.50
3
10
20.74±1.49
20.74
9.63
4.79
4
15
37.74±1.65
37.74
15.65
8.64
5
30
53.90±0.96
53.90
30.15
12.75
6
45
67.73±0.77
67.73
39.46
17.14
7
60
80.82±0.96
80.82
47.65
24.57
*All the values are expressed as mean ± SD, n=3.
100
90
80
70
60
50
40
30
20
10
0
F5
0 5 10 15 30 45 60
Time in minutes
Fig.9.21: Dissolution profile of formulation F5
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 84
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.18: Dissolution profile of formulation F6
S. No.
Time (min)
DR* (%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
9.48±1.22
9.48
5.71
2.50
3
10
20.83±1.89
20.83
11.04
4.82
4
15
36.84±1.55
36.84
16.59
7.69
5
30
53.31±1.72
53.31
29.56
12.62
6
45
61.44±0.91
61.44
38.83
17.58
7
60
68.16±2.17
68.16
45.76
20.43
*All the values are expressed as mean ± SD, n=3.
100
90
80
70
60
50
40
30
20
10
0
F6
0 5 10 15 30 45 60
Time in minutes
Fig.9.22: Dissolution profile of formulation F6
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 85
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.19: Dissolution profile of formulation F7
S. No. Time (min)
DR*
(%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
10.38±0.44
10.38
5.00
2.50
3
10
26.59±0.89
26.59
10.69
5.30
4
15
36.85±1.41
36.85
16.82
7.88
5
30
56.59±1.57
56.59
31.44
13.35
6
45
68.38±1.26
68.38
41.97
17.78
7
60
77.47±0.80
77.47
49.89
21.56
*All the values are expressed as mean ± SD, n=3.
100
90
80
70
60
50
40
30
20
10
0
F7
0 5 10 15 30 45 60
Time in minutes
Fig.9.23: Dissolution profile of formulation F7
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 86
Cu
mu
lati
ve %
dru
g re
leas
e
Table 9.20: Dissolution profile of formulation F8
S. No.
Time (min)
DR*
(%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
11.63±0.96
11.63
5.71
2.50
3
10
24.79±0.89
24.79
11.39
4.99
4
15
37.49±1.19
37.49
17.53
7.87
5
30
55.27±0.89
55.27
32.50
13.21
6
45
70.71±1.06
70.71
42.68
16.75
7
60
83.68±0.86
83.68
50.95
23.40
*All the values are expressed as mean ± SD, n=3.
100
90
80
70
60
50
40
30
20
10
0
F8
0 5 10 15 30 45 60
Time in minutes
Fig.9.24: Dissolution profile of formulation F8
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 87
Table 9.21: Dissolution profile of formulation F9
S. No.
Time (min)
DR*
(%)
Amount
(mg)
% DE
MDT
1
0
0.00
0.00
0.00
0.00
2
5
7.74±1.33
7.74
2.88
2.50
3
10
19.82±1.20
19.82
7.15
5.81
4
15
31.18±0.96
31.18
12.82
8.84
5
30
48.63±1.17
48.63
26.26
13.65
6
45
63.52±1.65
63.52
36.39
19.86
7
60
73.31±1.79
73.31
44.82
24.17
*All the values are expressed as mean ± SD, n=3.
Fig.9.25: Dissolution profile of formulation F9
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 88
Cu
mu
lati
ve %
dru
g rl
eas
e
Fig.9.26: Dissolution profile of inclusion complex (kneading method)
containing various ratio i.e. 1:1, 1:2, 1:3
100
90
80
70
60
50
40
30
20
10
0
F4
F5
F6
0 5 10 15 30 45 60
Time in minutes
Fig.9.27: Dissolution profile of inclusion complex (common solvent method)
containing various ratio i.e. 1:1, 1:2, 1:3
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 89
Cu
mu
lati
ve %
dru
g rl
eas
e
100
90
80
70
60
50
40
30
20
10
0
F7
F8
F9
0 5 10 15 30 45 60
Time in minutes
Fig.9.28: Dissolution profile of inclusion complex (physical mixture)
containing various ratio i.e. 1:1, 1:2, 1:3
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 90
Table 9.22: Comparative dissolution profile of all formulations
Time
in
(min)
Percentage drug release (%)
F1
F2
F3
F4
F5
F6
F7
F8
F9
0
0
0
0
0
0
0
0
0
0
5 8.25±
0.81
26.55±
0.80 8.27±
1.21
11.89±
1.33
10.45±
1.17
9.48±1.
22
10.38±
0.44
11.63±
0.96
7.74±
1.33
10 20.48±
1.22
43.33±
1.05
25.46±
0.67
24.68±
0.89
20.74±
1.49
20.83±
1.89
26.59±
0.89
24.79±
0.89
19.82
±1.20
15 33.86±
1.61
55.55±
1.92
32.83±
0.30
32.37±
1.25
37.74±
1.65
36.84±
1.55
36.85±
1.49
37.49±
1.19
31.18
±0.96
30 57.26±
0.33
85.18±
1.20
52.28±
0.22
51.09±
1.53
53.90±
0.96
53.31±
1.72
56.59±
1.57
55.27±
0.89
48.63
±1.17
45 75.09±
1.63
93.99±
0.29
65.89±
0.89
61.28±
1.40
67.73±
0.77
61.44±
0.91
68.38±
1.26
70.71±
1.06
63.52
±1.64
60 83.37±
1.81
97.54±
0.89
75.09±
1.49
72.85±
1.97
80.82±
0.96
68.16±
2.17
77.47±
0.80
83.68±
0.86
73.31
±1.79
*All the values are expressed as mean ± SD, n=3.
Fig.9.29: Comparative dissolution profile of all formulations
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 91
Fig.9.29: Comparative dissolution profile of all formulations
Fig. 9.30: Comparative dissolution profile of drug content, marketed
formulation and formulation F2
The dissolution studies revealed that all the formulations showed and
increased rate the complex prepared by kneading method was found to yield a
complex of higher rate of dissolution over common solvent and physical mixture
method. The results of the dissolution study indicates that formulation F2 prepared by
kneading method released to the maximum amount of drug and gave maximum
percentage of drug release was above 97.54 %±0.89 within hour when compared to
all other formulations.
The dissolution study of all the formulations was showed in Table 9.17-9.21
and Fig. 9.17-9.25.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 92
9.9. Stability Studies:
After storage the formulation F2 was analyzed for various parameters, results
are showed in Table 9.23.
Table 9.23: Stability study of formulation F2 of inclusion complex Aceclofenac at
room temperature 25° C ± 2° C / 60 % RH ± 5 % and accelerated temperature 40° C
± 2° C / 75 % RH ± 5 %.
Parameter
Initial
At 25° C ± 2° C / 60 %
RH±5 %
At 40° C ± 2° C / 75 %
RH ± 5 %.
1st
Month
2nd
Month
3rd
Month
1st
Month
2nd
Month
3rd
Month
Drug
content
(%)
99.69±
1.20
99.31±
1.54
99.20±
0.32
99.06±
0.28
99.54±
0.52
99.17±
0.35
99.02±
0.41
In vitro
drug
release at
end of 60
minute.
97.54±
0.89
97.39±
0.19
97.07±
0.98
96.81±
0.68
96.62±
1.11
96.35±
0.32
96.11±
1.01
*All the values are expressed as mean ± SE, n=3.
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 93
Fig.9.31: Comparisons of drug content before and after stability period at room
temperature (25° C ± 2° C / 60 % RH ± 5 %)
Fig.9.32: Comparisons of in vitro cumulative % drug release before and after stability
period at room temperature (25° C ± 2° C / 60 % RH ± 5 %)
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 94
Fig.9.33: Comparisons of drug content before and after stability period at accelerated
temperature (40° C ± 2° C / 75 % RH ± 5 %)
Fig.9.34: Comparisons of in vitro cumulative % drug release before and after
stability period at accelerated temperature (40° C ± 2° C / 75 % RH ± 5 %)
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 95
Fig.9.35: Comparisons of drug content before and after stability period at room
temperature and accelerated temperature
Fig.9.36: Comparisons of in vitro cumulative % drug release before and after stability
period at room temperature and accelerated temperature
Inclusion Complexes of Aceclofenac Results and Discussion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 96
The complex prepared by kneading method in the molar ratio of 1:2
(formulation F2) was found to exhibit a better stability under certain storage
condition.
There were no major differences between the evaluated parameters like drug
content, in vitro dissolutions and all the results were acceptable limits.
SUMMARY
AND
CONCLUSION
Inclusion Complexes of Aceclofenac Summary and Conclusion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 97
10. SUMMARY AND CONCLUSION
Among the emerging new chemical entities, most are poorly water soluble
drugs putting impact on their bioavailability and therapeutic effect. The solubility
enhancement techniques also play an important role in getting the excellent
dissolution properties of poorly soluble drugs. Successful improvement in aqueous
solubility is mainly depends on the method which we choose. Inclusion complex with
β-cyclodextrins is the most attractive technique to enhance aqueous solubility of
poorly soluble drugs. β-cyclodextrins, act as the useful solubilizer enabling both
solid/liquid oral and parenteral dosage forms. Solid binary system of drug and β-
cyclodextrins are capable to modify the physicochemical properties of drugs such as
solubility, particle size, crystal habit, thermal behavior, and there by forming a highly
water soluble amorphous forms. The β-cyclodextrins, due to their extreme high
aqueous solubility, they became capable to enhance the dissolution rate and bio-
availability of the poorly soluble drugs. The permeation of insoluble drugs through
various biological membranes can also be enhanced by preparing drug- β-
cyclodextrins inclusion compounds.
The present study was aimed to improving the dissolution of poorly soluble
drug Aceclofenac, being a drug acidic in nature shows a poor solubility in biological
fluids. Hence, an attempt was made to prepare β-cyclodextrins inclusion complex of
Aceclofenac by different techniques.
Literature survey revealed that cyclodextrin has been extensively study to
improve solubility, dissolution rate of various drugs. Due to its less price and easy
Inclusion Complexes of Aceclofenac Summary and Conclusion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 98
availability, β-cyclodextrin is widely used. It is a less toxic among all other
derivatives of cyclodextrins.
The preliminary work was studied by performing preformulation studies. The
identification drugs was determined by conducting the experiment on melting point,
solubility, loss on drying, FTIR and assay. The data obtained from these studies
indicated that the drugs are confirmed as Aceclofenac.
β-cyclodextrin inclusion complex were prepared by different methods like
kneading, common solvent and physical mixture in the ratio of 1:1, 1:2 and 1:3.
The prepared inclusion complex characterised by various analytical techniques
like UV spectrophotometer, Fourier Transform Infrared Spectroscopy, Differential
Scanning Calorimetry and X-ray diffraction studies.
The phase solubility studies were carried out according to the method Higuchi
and Connors. It revealed that the solubility of Aceclofenac increased linearly with the
increase in concentration of β-cyclodextrin.
The UV spectra of Aceclofenac and inclusion complex in the Methanol were
studied to know maximum absorbance (λmax). There was no shift in the λmax of
Aceclofenac in the presence of β-cyclodextrin.
Fourier Transform Infrared Spectroscopy and Differential Scanning
Calorimetry indicated the formation of true inclusion complexes of Aceclofenac with
β-cyclodextrin in 1:2 ratio prepared by kneading method.
The X-ray diffraction pattern confirmed the formation of complex with β-
cyclodextrin and the substance exhibits amorphous form.
The drug content of formulation F2 was found to be official limit.
Inclusion Complexes of Aceclofenac Summary and Conclusion
Adhiparasakthi college of pharmacy, Melmaruvathur. Page 99
From the dissolution profiles of inclusion complex prepared by kneading
method concludes that β-cyclodextrin is more meaningful for the enhancement of
solubility and dissolution rate of Aceclofenac.
The inclusion complex prepared by kneading method revealed that more stable
in the molar ratio of 1:2 at the end of three months.
So, it can be concluded that inclusion complex of Aceclofenac with β-
cyclodextrin could be prepared successfully by kneading method in a molar ratio of
1:2.
It can be concluded that an inclusion complex of Aceclofenac with β-
cyclodextrin could be prepared successfully by kneading in a molar ratio of 1:2 and
this was confirmed by phase solubility studies, UV spectrophotometry, Fourier
Transform Infrared Spectroscopy, Differential Scanning Calorimetry, X-ray
diffraction, drug content estimation, in vitro dissolution study and stability studies.
From the overall studies formulation F2 considered as best formulation.
FUTURE PROSPECTUS
Inclusion Complexes of Aceclofenac Future Prospectus
Adhiparasakthi College of Pharmacy, Melmaruvathur. Page 100
11. FUTURE PROSPECTUS
In the present work summarized and concluded the β-cyclodextrin inclusion
complex of Aceclofenac were prepared successfully by kneading method and it was
characterised by various analytical techniques. In the present investigation in vitro
characterisation was performed. Along with this study there is need of In vivo studies to
set up the in vitro – in vivo correlation for the better efficacy of Aceclofenac in the
treatment of rheumatoid arthritis with reducing gastrointestinal side effects.
Long term stability to be performed in future as per the ICH guidelines.
The best inclusion complex of Aceclofenac was chosen in future for
manufacturing of tablet formulation and evaluated for in vitro and in vivo performance.
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